The 5Th Workshop in the Nordic Arabidopsis Network

The 5th Workshop in the Nordic Arabidopsis Network:

“Arabidopsis as a Tool in Molecular Breeding”

Programme – Abstracts – Participants

Tune Landboskole, Denmark

October 13-15, 2005

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

Organizers: • Hans Thordal-Christensen Dept. of Agricultural Sciences The Royal Veterinary and Agricultural University Thorvaldsensvej 40 1871 Frederiksberg C Copenhagen, Denmark Tel: +45 35 28 34 43 Fax: +45 35 28 34 68 E-mail: [email protected]

• Jaakko Kangasjärvi Plant Biology, Dept. of Biological and Environmental Sciences University of Helsinki P.O. Box 56 00014 Helsinki Finland Tel: + 358 9 191 59444 Fax: + 358 9 191 59552 E-mail: [email protected]

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

Thursday, October 13

12.00 Registration at Tune Landboskole

12.30 Lunch & Setting-up posters

13.30 Welcome Hans Thordal-Christensen

13.35 Develop- Chairperson: Jaakko Kangasjärvi ment 13.35: Paul Grini - Gametophytic parental effects and seed I development in Arabidopsis 14.00: Vibeke Alm - An Arabidopsis SET domain protein with possible involvement in development of reproductional organs 14.15: Ellen D. Andersen - Genetic analysis of gametophytic parental effect mutants in Arabidopsis 14.30: Kim Andresen - Functional investigations of the Arabidopsis genes AtMBD8 and AtMBD11 encoding methyl- CpG-binding domain proteins. 14.45: Lars Nilsson - TERMINAL FLOWER2, the Arabidopsis HP1 homologue, a gene involved in several processes during plant development

15.00 Coffee

15.30 Invited Chairperson: John Mundy lecture 15.30: Dr. Jan Chojecki - Technology transfer and Arabidopsis

Develop- Chairperson: John Mundy ment 16.10: Eva Söderman - Homeodomain leucine zipper class I II genes in Arabidopsis: expression patterns and phylogenetic relationships 16.35: Henrik Johansson - Identification of downstream target genes of the HD-Zip class I transcription factors ATHB7 and ATHB12 in arabidopsis thaliana 16.50: Chamari Hettiarachchi - Characterization of HY5 interacting B-box proteins that regulate plant growth and development 17.05: Anna Honkanen - Genetic analysis of phloem development and differentiation in Arabidiopsis

18.30 Dinner

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

Friday, October 14 8.30 Invited Chairperson: Atle Bones lecture 8.30: Dr. Jian-Kang Zhu - Understanding and improving salt and other abiotic stress tolerance

Environ- Chairperson: Atle Bones mental 9.00: Annikki Welling - CBF transcription factors in birch cold stress acclimation 9.15: Saijaliisa Kangasjärvi - Role of reactive oxygen species in the regulation of light-harvesting complex II phosphorylation in chloroplasts 9.30: Hannes Kollist - Ozone-induced transient stomatal closure is absent in Arabidopsis mutants abi1, abi2 and rcd3

10.00 Coffee

10.30 Pumps Chairperson: Hans Thordal-Christensen and 10.30: Anja Fuglsang - Negative regulation of the plasma channels membrane H+-ATPase: protein kinase PKS5 action is dependent on a calcium binding protein and disrupts interaction of an activating 14-3-3 protein with the proton pump 10.55: Thomas Jahn - Ammonium: The paradox plant nutrient 11.20: Gerd-Patrick Bienert - New functions: two candidate aquaporins suggested to be involved in the transport of H2O2 11.35: Magnus Alsterfjord - 14-3-3 and H+-ATPase isoforms associated with the Arabidopsis leaf plasma membrane

12.30 Lunch

13.30 Poster viewing

15.00 Coffee

15.30 Breeding Chairperson: Hans Thordal-Christensen 15.30: Lene Olsen - The role of Dek1, Cr4 and Sal1 in cereal and Arabidopsis development 15.45: Marcus Bräutigam - Development of a Scandinavian winter oat by molecular breeding and transgenic techniques 16.00: John Einset - Determinants of chilling tolerance in Arabidopsis: How we can apply our knowledge to the selection of landscape plants for Nordic regions 16.15: Erika Groth - The evolution of reproductive organ development in seed plants 16.30: Carsten Meier - At at land mines

18.30 Dinner

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

Saturday, October 15 8.30 Invited Chairperson: Stanislaw Karpinski lecture 8.30: Dr. Jian-Kang Zhu - Role of microRNAs and siRNAs in abiotic stress responses

Disease Chairperson: Stanislaw Karpinski resistance 9.00: Tapio Palva - Interacting pathways in Arabidopsis defense I signaling 9.25: Ziguo Zhang - Syntaxins suppress salicylic acid-mediated defence 9.50: Frederikke Malinovski - Screening for suppressors of Arabidopsis acd11

10.10 Coffee

10.40 Disease Chairperson: Stanislaw Karpinski resistance 10.40: Christina Dixelius - Defence mechanisms in Arabidopsis II to Leptosphaeria maculans 11.05: David B. Collinge - Barley genomics and plant defence responses 11.30: Pernille Olsen - Promoters for genes up-regulated in powdery mildew infected plant cells 11.45: Carl Gunnar Fossdal - Defense reactions in norway spruce toward the pathogenic root-rot causing fungus Heterobasidion annosum 12.00: Mari-Anne Newman - Lipo-oligosaccharide of Xanthomonas campestris triggers innate immune response in Arabidopsis

12.30 Lunch

13.30 Disease Chairperson: Jaakko Kangasjärvi resistance 13.30: Erik Andreasson - Characterisation of stress-related MAP & kinase substrates in Arabidopsis Environ- 13.55: Stansilaw Karpinski - On the role of excess excitation mental energy in regulation of plant defence responses; holistic analysis stress of plants’ stress signalling network 14.20: Annavera De Felice - Diurnal changes in myrosinase- glucosinolate system: effects of photoperiod and temperature 14.35: Tarja Kariola - ERD15 modulates disease resistance and freezing tolerance in Arabidopsis

14.50: Closing of workshop and network

15.00 Coffee

15.30 Departure

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

INVITED LECTURES

ABSTRACTS

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

TECHNOLOGY TRANSFER AND ARABIDOPSIS

Jan Chojecki

PBL, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK

Arabidopsis serves as an extremely important platform for plant science and has much to teach about fundamental plant biology and developmental processes. However, it must be recognised that Arabidopsis was selected as a research model because of the convenience of its genetics and generation cycle, rather than because of its phenotypic relevance to economically important plants. Although the last two decades of genomic research has underlined the strength of the genetic relationship amongst all plant species, discoveries made in Arabidopsis are still a very long way from application in economically relevant plants.

Understanding gained from Arabidopsis research might be expected (hoped) to be transferred to applied use in any of three main ways: genetic manipulation of plant traits via transgenic (GM) routes; enhanced use of existing (or induced) genetic variation within germplasm pools; or enhanced crop management practices gained from understanding of how plants interact with the biotic and abiotic environment. The presentation will address why the potential for exploiting these channels from Arabidopsis is not readily being fulfilled - for a number of reasons including practical and technical barriers, research culture, the conditions in the plant technology industry, and intellectual property considerations.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

UNDERSTANDING AND IMPROVING SALT AND OTHER ABIOTIC STRESS TOLERANCE

Jian-Kang Zhu

Institute for Integrative Genome Biology, and Department of Botany and Plant Sciences, 2150 Batchelor Hall, University of California, Riverside, CA 92521, USA

Salt and other related abiotic stresses have multiple effects on plants including ionic, osmotic and oxidative imbalances. Plants have evolved multiple response pathways to cope with these stresses. Various genes in these pathways have been used to improve plant abiotic stress tolerance. Recent progress in our understanding of some of the abiotic stress response pathways and strategies to improve plant tolerance will be presented.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

ROLE OF MIRNAS AND SIRNAS IN ABIOTIC STRESS RESPONSES

Jian-Kang Zhu

Institute for Integrative Genome Biology, and Department of Botany and Plant Sciences, 2150 Batchelor Hall, University of California, Riverside, CA 92521, USA

Small non-coding RNAs ranging in size between 20 and 24 nucleotides are important regulators of mRNA degradation, translational repression, and chromatin modification. These small RNAs can be broadly classified as miRNAs (microRNAs) and siRNAs (short interfering RNAs) based on their biogenesis. We found that the expression of some miRNAs and siRNAs in Arabidopsis plants are regulated by abiotic stresses such as drought, soil salinity and cold temperatures. Data on the functional analysis of several miRNAs and siRNAs using transgenic plants will be presented to support the regulatory roles of small RNAs in plant adaptation to abiotic stresses.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

ORAL PRESENTATIONS

ABSTRACTS

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

GAMETOPHYTIC PARENTAL EFFECTS AND SEED DEVELOP- MENT IN ARABIDOPSIS

Moritz Nowack2, Nirma Skrbo1, Ellen D. Andersen1, Martin Hülskamp4, Gerd Jürgens3, Arp Schnittger2, Reidunn B. Aalen1 and Paul E. Grini1

1Molecular Biosciences Department, University of Oslo, Oslo, Norway 2University Group at the Max-Planck-Institute for Plant Breeding Research, Cologne, Germany 3ZMBP, Developmental Genetics Department, University of Tübingen, Tübingen, Germany 4Botanical Institute III, University of Cologne, Cologne, Germany.

Seed development requires a coordinated interplay of embryo, endosperm and the maternal seed coat. What roles gametophytic parental (maternal and paternal) factors play in this process is not clear. We have performed various screens to identify haplo-phase specific genes required in the gametophytic phase, or required in a gametophytic parental effect specific manner for embryo and endosperm development Here we present an outline of these screens. Our main focus is to identify and characterize the action of genes that have a parental effect on seed development. In the gametophytic maternal-effect capulet (cap) mutants, both embryo and endosperm development is arrested at early stages. The cap mutant phenotypes are not rescued by wild-type pollen and removal of silencing barriers from the paternal genome by METHYL TRANSFERASE1 antisense transgene expression or by mutation in the DECREASE IN DNA METHYLATION1 (DDM1) gene also fails to restore seed development. The mutants display no autonomous seed development and were epistatic to medea/ fertilisation-independent-seed1 (fis1) in both autonomous and sexual endosperm development. Embryo and endosperm specific GUS and GFP markers show ectopic expression in cap mutant endosperms. Molecular characterization of the CAPULET genes is in progress to verify whether the CAPULETs represent novel maternal functions supplied by the female gametophyte that are required for embryo and endosperm development. As an additional tool to dissect the involvement of maternal and paternal gene programs in seed development we explore the use of a paternal effect cdka;1 mutant line where mutant pollen induce embryo and endosperm developmental arrest after fertilization. In cdka;1 pollen the second mitotic division of the generative cell is not made, and a single generative-like cell preferentially fertilizes the egg cell, leading to partial development of both embryo and (unfertilized) endosperm. We observed exclusive fertilization of the egg cell by cdka;1 sperm cells. Moreover, we show that unfertilized endosperm develops, revealing a previously unrecognized positive signal from the fertilization of the egg cell initiating proliferation of the central cell. The cdka;1 mutant line allows genetic dissection of parental contribution to the fertilization product and a novel tool in the seach for factors required for autonomous seed development.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

AN ARABIDOPSIS SET DOMAIN PROTEIN WITH POSSIBLE INVOLVEMENT IN DEVELOPMENT OF REPRODUCTIONAL ORGANS

Vibeke Alm, Paul E. Grini, and Reidunn B. Aalen

The Arabidopsis Group, Program for Molecular Genetics, Department of Molecular Biosciences, University of Oslo, Postboks 1041, Blindern, 0316 Oslo, Norway

Plant developmental patterning and flowering time are important processes in which information needed to be transformed from an earlier event to a later one has to extend the operating area of a gene, which is limited to a few cells, and its persistency, which is limited to shorter distances. Fixation of the transcriptional state involving epigenetic changes is one solution to ensure information from earlier events to be transformed stably through somatic cell development (mitosis), and also reversibility, so that info accruing through somatic development can be erased by the onset of each new generation (through meiosis). One group of proteins involved in epigenetic changes is the SET domain proteins, which contains the 130-160 amino acid long SET domains. The SET domain are named after three proteins which were originally identified in Drosophila; supressor of variegation (SU(VAR)3–9), enhancer of zeste (E(Z)) and trithorax (TRX). SU(VAR)3–9 is a suppressor of Position Effect Variegation (PEV), E(Z) belongs to the polycomb group (PcG) proteins, which are involved in stable transcriptional repression, whereas TRX is a member of trithorax group (TrG) proteins, which are involved in stable transcriptional activation. The SET domain proteins are involved in multimeric protein–protein interactions and share distinctive amino acid motifs like the SET domain, but also other domains like the chromo domain, CXC domain and the PHD finger. The SET domain catalyzes histone methyl transferase activity which is the epigenetic change determining the transcriptional activity of the target of the complex. One of the at least 29 active SET domain genes in Arabidopsis thaliana reported in Baumbush and Thorstensen et al. (Nucleic Acids Research, 2001) has three independent SALK lines showing the same pleiotropic phenotype including reduced fertilization. Morphological analysis and specific in situ expression pattern indicate involvement of this protein in seed development, as does cDNA microarray results of one of the SALK T-DNA lines of this gene. Proper seed development is crucial in agronomic important plants, and this study will hopefully gain insight into part of this process at an epigenetic level.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

GENETIC ANALYSIS OF GAMETOPHYTIC PARENTAL EFFECT MUTANTS IN ARABIDOPSIS

Ellen D. Andersen1, Gerd Jürgens2, Martin Hülskamp3, Reidunn B. Aalen1 and Paul E. Grini1 1Molecular Biosciences Department, University of Oslo, Oslo, Norway 2ZMBP, Developmental Genetics Department, University of Tübingen, Tübingen, Germany 3 Botanical Institute III, University of Cologne, Cologne, Germany The life cycle of plants comprises two alternating generations, the diploid sporophyte and the haploid gametophyte. In higher plants, the main plant body is the sporophyte, and the gametophytes are reduced to few-celled structures that develop from postmeiotic cells inside the reproductive tissues of the sporophytic plant. Mechanisms underlying development and function of higher- plant gametophytes are still poorly understood, which is mainly due to the technical difficulties of identifying relevant gene functions by mutant phenotypes. Screening a collection of 1,300 Ds gene-trap insertion lines, we isolated nine gametophytic mutants detectable by their reduced transmission of the Ds transposon-borne kanamycin resistance marker. In reciprocal crosses, all lines showed reduced resistance marker transmission through the pollen. Transmission through the embryo sac was also affected in four of the nine lines. Morphological characterization of the mutants revealed gametophytic defects supporting the genetic analysis. Pollen development was arrested at various stages after the first mitosis in most lines. Two lines were defective in the asymmetric division, producing ectopic walls inside the pollen grain. With one exception, female-gametophytic defects were developmental arrests at different stages of embryo sac development. In the line 416/cap3, embryo and endosperm development was affected in a gametophytic maternal-effect manner, and the mutant phenotype was not rescued by wild-type pollen or hypomethylated pollen from a METHYLTRANSFERASE1antisense transgenic line. In order to reveal the molecular nature of the genes affected in the isolated lines we use a modified splinkerette cloning system to identify the regions flanking the DsG transposons. The identified genes present different classes of proteins, including a putative C2H2 transcription factor, a member of the cellulose synthase family of proteins and a member of the DUF642 protein family. Further work is in progress and will be presented.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

FUNCTIONAL INVESTIGATIONS OF THE ARABIDOPSIS GENES AtMBD8 AND AtMBD11 ENCODING METHYL-CpG-BINDING DOMAIN PROTEINS.

Ellen Maryann Rosenhave, Kim Andresen, Anita Berg and Reidunn B. Aalen

The Arabidopsis Group, Program for Molecular Genetics, Department of Molecular Biosciences, University of Oslo, PO Box 1041 Blindern, 0316 Oslo

The genome of Arabidopsis thaliana contains twelve putative genes encoding proteins with domains similar to the methyl-CpG-binding (MBD) domain found in animals and other eukaryotes. In vertebrates, most MBD proteins seem to function at an epigenetic level of regulation, through the participation in transcriptional regulation through interactions with proteins involved in chromatin remodelling, such as histone deacetylases. Here we focus on genetic and molecular characterization of the AtMBD8 and AtMBD11 genes. We have, in our collection of T-DNA mutagenised lines in C24 background, an AtMBD8 mutant showing delayed flowering. We are currently in the process of identifying interacting partners to the AtMBD8 protein by yeast two-hybrid analysis. Experiments are also being conducted to investigate the role of the AtMBD11 gene in C24 and Columbia background, employing temporal and spatial expression analyses and studies of SALK insertion lines.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

TERMINAL FLOWER2, THE ARABIDOPSIS HP1 HOMOLOGUE, A GENE INVOLVED IN SEVERAL PROCESSES DURING PLANT DEVELOPMENT

Nilsson, Lars1; Landberg, Katarina1; Para, Alessia2 and Sundås Larsson, Annika1

1Department of Physiological Botany, Evolutionary Biology Centre, Uppsala University, Villavägen 6, SE-752 36 Uppsala, SWEDEN 2Cell Biology-ICND 216 , The Scripps Research Institute , 10550 N. Torrey Pines Road, La Jolla CA 92037, USA. E-mail: [email protected]

The Arabidopsis thaliana gene TERMINAL FLOWER2 (TFL2) encodes a protein containing a chromo (chromatin organization modifier) domain and a chromo shadow domain that characterizes the metazoan HETEROCHROMATIN PROTEIN1 (HP1) proteins. The Arabidopsis gene encodes a 445 amino acid protein compared to animal proteins of approximately 200 amino acids. These proteins are found to function in the regulation of gene expression as one of the components of chromatin structure. The tfl2 mutation gives rise to dwarfed plants that still show the wildtype proportions in all of the above ground parts of the plant. The rosette leaves are smaller and the lengths of the internodes are shorter, even though organisation and cell size of the vegetative meristem shows normal proportions. The mutated plant shows no differences from wild type phyllotaxy and root development and after producing approximately 20 flowers there is a termination of the main inflorescence axis which leads to a reduction in the apical dominance. Analysis have shown that the TFL2 mRNA is weakly expressed in all above ground tissues and at higher levels in shoot apical meristems, floral meristems and in young proliferating tissues. The pleiotropic phenotype of the mutant and the homology to mammalian genes suggest that the gene product is involved in the regulation of a wide range of processes during plant development. Regarding regulation at the level of chromatin we have found genetic interaction with factors earlier described to function in this process such as FASCIATA and TOPOISOMERASE1. With respect to the mutants early flowering phenotype it is shown that TFL2 is involved in the regulation of the transition to flowering in at least the autonomous and the long day pathway. We also show that TFL2 is involved in processes regulating photomorphogenesis.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

HOMEODOMAIN LEUCINE ZIPPER CLASS I GENES IN ARABIDOPSIS: EXPRESSION PATTERNS AND PHYLOGENETIC RELATIONSHIPS

Henriksson, Eva; Olsson, Anna; Johannesson, Henrik; Johansson, Henrik; Hanson, Johannes; Engström, Peter and Söderman, Eva.

Department of Physiological Botany, Evolutionary Biology Centre, University of Uppsala, Villavägen 6, SE-752 36 Uppsala, Sweden. [email protected]

Members of the homeodomain leucine zipper (HDZip) family of transcription factors are present in a wide range of plants, from mosses to higher plants, but not in other eukaryotes. The HDZip genes act in developmental processes, including vascular tissue and trichome development, and several of them have been suggested to be involved in the mediation of external signals to regulate plant growth. The Arabidopsis thaliana genome contains 47 HDZip genes, which based on sequence criteria have been grouped into four different classes; HDZip I-IV. In this report we present an overview of the class I HDZip genes in Arabidopsis. We describe their expression patterns, transcriptional regulation properties, duplication history and phylogeny. The phylogeny of HDZip class I genes is supported by data on the duplication history of the genes as well as the intron/exon patterning of the HDZip encoding motifs. The HDZip class I genes were found to be widely expressed and partly to have overlapping expression patterns at the organ level. Further, ABA or water deficit treatments and different light conditions affected the transcript levels of a majority of the HDZip I genes. Within the gene family, our data show examples of closely related HDZip genes with similarities in the function of the gene product, but a divergence in expression pattern. In addition, six HDZip class I proteins tested were found to be activators of gene expression. In conclusion, several HDZip I genes appear to regulate similar cellular processes, though in different organs or tissues and in response to different environmental signals.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

IDENTIFICATION OF DOWNSTREAM TARGET GENES OF THE HD- ZIP CLASS I TRANSCRIPTION FACTORS ATHB7 AND ATHB12 IN ARABIDOPSIS THALIANA.

Johansson, Henrik; Olsson, Anna; Övernäs, Elin; Engström, Peter and Söderman, Eva

Department of Physiological Botany, Evolutionary Biology Centre, Uppsala University, Villavägen 6, 752 36, Uppsala, Sweden.

The homeodomain leucine zipper (HDZip) proteins constitute a group of plant- specific transcription factors. The homeodomain of the proteins bind specific DNA-sequences, while the leucine-zipper mediates protein-protein dimerisation. The Arabidopsis thaliana genome contains 42 HDZip encoding genes, divided into four classes (I to IV). The class I HDZip protein encoding genes are in their turn divided into six subclasses (α to φ), based on sequence similarities, exon- intron patterning and traced duplication history. The expression levels of the two members of subclass γ, the paralogous genes ATHB7 (Arabidopsis thaliana homeobox 7) and ATHB12, increase in response to drought, salt and abscisic acid (ABA). ATHB7 and ATHB12 belong to an exclusive set of transcription factors always being induced during water-deficit conditions, indicating an important role in the plants stress response. Analyses of athb7 and athb12 single and double T-DNA insertion mutants show reduced ABA sensitivity in root elongation assays and phenotypic deviations from wild-type in stem and leaf growth. Transgenic plants with increased transcript levels of ATHB7 and/or ATHB12 show additive reductions of growth in stem and leaf as well as roots in response to ABA. Thus, we hypothesise that ATHB7 and ATHB12 are involved in growth regulation in stems, leaves and roots under water deficit conditions and that they may act redundantly to each other. To identify the molecular components involved in this regulation of growth, we are analysing the downstream gene repertoire of ATHB7 and ATHB12 using two different microarray-based approaches; (1) using an in-house manufactured cDNA-array representing 1304 Arabidopisis transcription factors, we are studying the downstream genes of an athb12 single mutant and the corresponding wild-type with and without ABA-treatment, thereby identifying transcriptional regulators dependant on the expression of ATHB12, (2) using the Affymetrix ATH1 GeneChip, we are studying the transcriptome of an athb7/athb12 double mutant (circumventing any redundancy effects between ATHB7 and ATHB12) and its corresponding wild-type with and without ABA-treatment. Results from these experiments will be presented at the workshop.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

CHARACTERIZATION OF HY5 INTERACTING B-BOX PROTEINS THAT REGULATE PLANT GROWTH AND DEVELOPMENT.

Hettiarachchi, Chamari1; Datta, Sourav1; Desai, Mintu1; Deng, Xing-Wang2 and Holm, Magnus1.

1) CMB-Molecular Biology, Gothenburg University, Gothenburg, Sweden. 2) MCDB Yale University, New Haven, CT, USA

Light is an important factor for plant growth and development and a dramatic example of light signaling can be seen during seedling development. A seedling that encounters light de-etiolates resulting in inhibition of hypocotyl elongation, promotion of cotyledon expansion and synthesis of number of pigments including chlorophyll and anthocyanin. De-etiolation is induced by wavelength specific photoreceptors in the nucleus and entails a dramatic transcriptional reprogramming. The bZIP transcriptional factor HY5, which acts downstream of several photoreceptors is a positive regulator of de-etiolation. In the dark, HY5 activity is negatively regulated by COP/DET/FUS mediated degradation of the HY5 protein. In addition to HY5, COP1 degrade the transcription factors HYH, LAF1 and HFR1 in the dark. We have identified four Arabidospsis B- box proteins in yeast two hybrid screens (STH, STO, STO1 and STO2) that interact with HY5, COP1 or both. I have isolated T-DNA insertion mutants in each of the four genes and phenotypic analysis of these mutants revealed pigmentation, hypocotyl and root phenotypes suggesting that the genes have positive roles in light and HY5 regulated processes. Furthermore, STH, STO and STO2 enhance the ability of HY5 to activate reporters for HY5 regulated genes in a transient protoplast assay. Thus I have genetic, functional and cell biological data suggesting that these B-box proteins positively regulate HY5 activity in light.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

GENETIC ANALYSIS OF PHLOEM DEVELOPMENT AND DIFFEREN-TIATION IN ARABIDIOPSIS

Honkanen, Anne; Carlsbecker, Annelie, Bonke, Martin, Lindgren, Ove, Tähtihärju, Sari, Tithamadee, Siripong and Ykä Helariutta

Institute of Biotechnology, Viikinkari 4, FIN-00014 University of Helsinki, Finland

We have shown that phloem development in the Arabidopsis root is established by a specific set of cell divisions. In the recessive mutant altered phloem development (apl) these cell divisions are disturbed and the phloem-pole cells differentiate xylem characteristics instead of phloem (Bonke et al., 2003, Nature 426:181). APL encodes a MYB coil-coiled transcription factor active specifically in protophloem and mature phloem cells. Ectopic APL expression results in inhibition of xylem development but not ectopic phloem development, indicating that APL is necessary, but not sufficient for phloem differentiation. To identify additional factors in phloem development we performed a genetic screen of an EMS-mutagenized companion cell-specific marker line. This resulted in the identification of a set of novel mutants with patterning and/or cell proliferation defects specific to the stele. Here we present the characterization of these mutants. In combination with a forward genetic approach we aim at identify factors acting up-and/or down-stream of APL in the regulation phloem development and differentiation in Arabidopsis.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

CBF TRANSCRIPTION FACTORS IN BIRCH COLD ACCLIMATION

Annikki Welling1, Tuula Puhakainen1 & Tapio Palva2

Department of Biological and Environmental Sciences, Plant Biology1, P.O.Box 65, Genetics2, FIN-00014 University of Helsinki, Finland

Cold acclimation of plants in response to low temperature (LT) requires changes in gene expression. Several of the LT-inducible genes contain an LT-responsive DNA regulatory element referred to as LTRE/DRE/CRT-element. In Arabidopsis three cold-inducible transcription activators that bind to this element has been identified, designated CBF1-3, and shown to play an important regulatory role in plant cold acclimation. Similarly to Arabidopsis, boreal zone trees can increase freezing tolerance (FT) in response to LT during growing season. However, maximum FT of these trees is achieved during overwintering, after growth cessation triggered by short daylength (SD) and subsequent exposure to low and freezing temperatures. To elucidate the molecular basis of cold tolerance, we explored the regulons involved in birch (Betula pendula Roth) cold acclimation. Birch dehydrin gene Bplti36 has been shown to be cold inducible and to contain several LTRE/DRE/CRT elements on its promoter. Reporter gene-promoter fusion analyses showed that Arabidopsis CBFs recognize the birch element causing an induction of the reporter gene, suggesting that birch has an operational CBF regulon. We identified several putative CBFs in birch EST libraries and chose two slightly different genes for functional studies. LT stimulus induced expression of birch CBF genes rapidly in growing birch leaves and in stems and buds of dormant, SD grown plants. The expression was downregulated after few hours of LT stimulus. Expression of the CBF target gene, Bplti36, was sequential to birch CBFs. Ectopic expression of birch CBFs in Arabidopsis led to a stunted growth and delayed flowering of transgenic plants. In addition, these plants showed constitutive expression of an endogenous CBF target gene, LTI78 and increased FT without LT stimulus. Taken together, our results suggest that studied birch genes indeed are CBFs and they are involved in cold acclimation of birch both during growing season and overwintering periods.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

ROLE OF REACTIVE OXYGEN SPECIES IN THE REGULATION OF LIGHT-HARVESTING COMPLEX II PHOSPHORYLATION IN CHLOROPLASTS

Saijaliisa Kangasjärvi, Minna Lintala, Anna Lepistö and Eevi Rintamäki

Department of Biology, University of Turku FIN-20014 Turku, Finland

The photosynthetic light reactions generate reducing equivalents and reactive oxygen species (ROS) that possess counteracting reducing and oxidizing effects on the reduction state of chloroplasts. Under stressful conditions, these reactive intermediates have a potential to damage the photosynthetic machinery, which may lead to reduction in the photosynthetic capacity, and ultimately in reduced growth and productivity.

Our present study focuses on the role of ROS and their scavenging enzymes in the complex regulation of chloroplast metabolism, and particularly the phosphorylation of Photosystem II light harvesting antenna (LHCII) proteins in vivo. Despite intensive research, the physiological role of LHCII protein phosphorylation is still under debate. The LHCII kinase is activated in light by reduction of cytochrome b6/f complex in thylakoid membrane. Furthermore, we have previously established novel regulatory mechanisms of LHCII protein phosphorylation, involving thiol-redox regulation and H2O2, both also being potential components in signalling pathways regulating chloroplast-dependent gene expression in the nucleus.

Chloroplast ascorbate peroxidases (APXs) reduce H2O2 to water using ascorbate as an electron donor, and thus play an important role in the complex detoxification systems for H2O2. We analyzed Arabidopsis thaliana t-apx and s-apx mutants deficient in the thylakoid-bound and stromal forms of APX, respectively. No significant differences in growth rate, leaf thickness, chlorophyll content or the level of LHCII protein phosphorylation were observed between the mutant and wild type lines after long-term growth under different light intensities. However, upon a shift of plants to different light regimes, the s-apx plants were able to maintain the LHCII kinase active at light intensities that induced inhibition of LHCII kinase activity in wild type plants. We propose that H2O2 in chloroplast stroma of s-apx plants is involved in the maintenance of LHCII protein phosphorylation under elevated light intensities in vivo. Finally, we suggest that the LHCII kinase, being under redox control on several levels, functions as a delicate sensor of imbalances in chloroplast redox state under environmental stresses, and is involved in the initiation of defence reactions and acclimation processes in plants.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

OZONE-INDUCED TRANSIENT STOMATAL CLOSURE IS ABSENT IN ARABIDOPSIS MUTANTS abi1, abi2 AND rcd3

Hannes, Kollist1,2; Triin, Kollist1,2; Bahtior, Rasulov3; Airi, Lamminmäki2; Hanne, Teittinen2; Vello, Oja3; Heikko, Rämma3; Katja, Hüve3; Olevi, Kull1; Heino, Moldau1; Jaakko, Kangasjärvi2

1 Institute of Botany and Ecology, University of Tartu, Lai 40, Tartu, Estonia. 2 Plant Biology, Department of Biological and Environmental Sciences, Viikki Biocenter, University of Helsinki, POB 56 (Viikinkaari 9), 00014 Helsinki, Finland. 3 Institute of Molecular and Cell Biology, University of Tartu, Riia str. 23, Tartu, Estonia

Ozone (O3), the predominant air pollutant, has shown to be useful tool to induce acute formation of reactive oxygen species (ROS) in plants and to identify molecular components regulating ROS induced processes in leaf cells. A number of ozone-sensitive Arabidopsis mutants has been identified and used to elucidate the molecular mechanisms of plant O3-sensitivity. Although the importance of stomata in controlling the flux of O3 into the leaf is widely recognized, mutant analysis to elucidate the molecular mechanisms involved in stomatal regulation in relation to O3-influx has not yet been sufficiently used. This is due to experimental difficulties with monitoring gas exchange of Arabidopis rosette during O3-exposure. We have developed a system for measuring gas exchange in Arabidopsis. It consists of eight whole-rosette O3 exposure/gas exchange units and enables the analysis of O3 uptake, CO2 assimilation and stomatal conductance in in real time. Acute exposure to 75-250 -1 nl l of O3 induced a rapid stomatal closure within 6-10 min in 11 out of 14 different Arabidopsis accessions tested, however, without affecting the net CO2 assimilation. Thereafter the stomatal conductance regained its initial value within an hour in spite of the continuing exposure to O3. In the abscisic acid insensitive mutants abi1, abi2, and in a novel O3-sensitive mutant, rcd3, the transient closure was absent. High correlation between stomatal conductance and membrane leakage suggests an important role for stomatal regulation in controlling O3 uptake and determining O3 sensitivity in Arabidopsis. The results are discussed on the background of current mechanistic models of reactive oxygen species-, Ca2+- and anion-mediated stomatal responses.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

NEGATIVE REGULATION OF THE PLASMA MEMBRANE H+- ATPase: PROTEIN KINASE PKS5 ACTION IS DEPENDENT ON A CALCIUM BINDING PROTEIN AND DISRUPTS INTERACTION OF AN ACTIVATING 14-3-3 PROTEIN WITH THE PROTON PUMP.

Fuglsang, Anja T. 2§, Guo, Yan1§, Qiu, Q.1, Son,C.1, Schumaker, K.S.1, Palmgren, M.2, J-K. Zhu1,3

1Department of Plant Sciences, University of Arizona, Tucson, AZ85721; 2Department of Plant Biology, Royal Veterinary and Agricultural University, KVL, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. 3Current address: Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, 2150 Batchelor Hall, University of California, Riverside, CA 92521

Regulation of cellular pH is an important part of plant responses to several hormonal and environmental cues including auxin, blue light and fungal elicitors. However, little is known about the signaling components that mediate cellular pH homeostasis in plants. Here we report that an Arabidopsis serine/threonine protein kinase, PKS5, is a critical regulator of cellular pH homeostasis and plant responses to alkaline conditions. Loss-of-function pks5 mutant plants are more tolerant of high external pH. PKS5 negatively regulates the activity of plasma membrane H+-ATPase, and it can phosphorylate the H+- ATPase AHA2 at a novel site, Ser931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between H+-ATPase and an activating 14-3-3 protein. We show that PKS5 interacts with a calcium-binding protein SCaBP1, and that high external pH can trigger an increase in the concentration of cytosolic free calcium. These results suggest that PKS5 is part of a calciumsignaling pathway mediating H+-ATPase regulation.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

AMMONIUM: THE PARADOX PLANT NUTRIENT

Jahn, Thomas P.; Møller, Anders L.B.;ten Hoopen, Floor; Bienert, Gerd- Patrick; Hegelund, Josefine N.; Schüssler, Désirée and Schjoerring, Jan K.

Plant and Soil Laboratory, Department of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark e-mail: [email protected]; fax.: +45 35 28 34 60

+ Ammonium (NH4 / NH3) is a central intermediate for nitrogen assimilation in + both plants and animals. However, elevated NH4 is potentially toxic and has been linked to NH3 volatilization from plant foliage and loss in nitrogen use + efficiency. We have studied on the molecular level the transport of NH4 / NH3 across membranes to better understand ammonium toxicity and nitrogen use efficiency. Using heterologous expression in yeast and oocytes, selected members of the aquaporin super family have been identified able to transport NH3 hence aquaammoniaporins (Jahn et al. 2004, FEBS 574, 31-36). Aquaammoniaporins have been localized in the tonoplast, the peribacteroid membrane, the chloroplast inner membrane and the plasma membrane. The observation of facilitated diffusion of NH3 was initially puzzling, since a large body of + physiological data in the literature suggest that NH4 is the main form + transported across the plant plasma membrane. The presence of both, NH4 uptake and NH3 release at the plasma membrane would be problematic, since the transport may result in the collapse of the proton motive force or the membrane potential. Here we present data from yeast and Arabidopsis, strongly suggesting that some + + K transporters can transport NH4 and that this transport under high ammonium and low potassium supply is toxic to both yeast and Arabidopsis. + + Our data suggest that transport of NH4 through K transporters may represent a major thread leading to ammonium toxicity and reduced nitrogen use efficiency. We are now in the process of characterizing plants with altered expression levels of various ammonium transporters.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

NEW FUNCTIONS: TWO CANDIDATE AQUAPORINS SUGGESTED TO BE INVOLVED IN THE TRANSPORT OF H2O2

Bienert, Gerd-Patrick; Schüssler, Désirée; Schjoerring, Jan K. and Jahn, Thomas P.

Hydrogen peroxide (H2O2) belongs to the reactive oxygen species (ROS), known as oxidants that can react with various cellular targets thereby causing cell damage or even cell death. On the other hand, recent work from various laboratories has demonstrated that H2O2 functions as a signalling molecule controlling different essential processes in plants and mammals. Because of these opposing functions the cellular level of H2O2 is likely to be subjected to tight regulation via mechanisms such as production, distribution and removal. Substantial progress has been done exploring the formation and scavenging of H2O2 whereas very little is known about how this signal molecule is transported from its site of origin to the place of action or detoxification. As H2O2 is thought to be freely diffusible across membranes, diffusion was believed to be the exclusive way of transport. Contrary to this assumption recent studies pointed out that some membrane are only weakly permeable to H2O2. H2O2 transport across those membranes may thus be facilitated by either a change in the lipid composition or via facilitated diffusion through channel proteins. Using various yeast mutants (∆tsa1,2; ∆yap1; ∆skn7) compared to wild type with different sensitivities to exposure to H2O2, we identified aquaporins that, when expressed in yeast, increased the sensitivity to externally applied H2O2. A screening was performed using a total of 25 aquaporins homologues from plants and mammals. Yeast mutants transformed with either HsAQP8 or AtTIP1;1 increased the sensitivity to H2O2 by a factor of up to 10 fold suggesting a function in H2O2 transport. A more direct in vitro transport assays using H2O2 sensitive fluorescent probes and stopped flow fluorometry is currently under investigation. Our results suggest that selected aquaporins play a role in the regulation of H2O2 distribution and may be important players in oxidative stress and apoptosis.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

14-3-3 AND H+-ATPASE ISOFORMS ASSOCIATED WITH THE ARABIDOPSIS LEAF PLASMA MEMBRANE

Alsterfjord, Magnus1; Sehnke, Paul2; Arkell, Annika1; Müller, Markus3; Ferl, Robert2; Larsson, Christer and Sommarin, Marianne1,3

1Dept of Plant Biochemistry, Lund University, Box 124, SE-221 00, Lund, Sweden 2Dept of Horticultural Sciences, University of Florida, P.O. Box 110690, Gainesville, FL, USA, 32611-0690 3Dept of Plant Physiology, Umeå Plant Science Center, Umeå University, SE- 901 87, Umeå, Sweden

The plant plasma membrane is energized by a family of H+ pumping ATPases, which produce an electrochemical gradient across the membrane. There are 12 genes encoding plasma membrane H+-ATPases in the Arabidopsis genome and at least 11 of them are expressed. These H+-ATPases are activated by phosphorylation of the penultimate Thr in the C terminus and concomitant binding of 14-3-3 protein, which displaces a C-terminal autoinhibitory domain. 14-3-3s constitute a family of eukaryotic proteins that are key regulators of a large number of processes. 14-3-3s function as dimers and bind to particular (often phosphorylated) motifs in their target proteins. There are 15 genes encoding 14-3-3s in the Arabidopsis genome and at least 13 of them are expressed. In plants, 14-3-3s regulate major metabolic pathways by regulating enzymes such as nitrate reductase, sucrose phosphate synthase, starch synthase, and plasma membrane H+-ATPase. Using 14-3-3 overlays we now show that of all twelve 14-3-3 isoforms tested, all are able to bind to the H+-ATPase isoforms expressed in the Arabidopsis leaf plasma membrane. However, using antibodies specific to 9 of these 14-3-3 isoforms we show that only a limited number of these are expressed in the leaf and bind to the plasma membrane. Similarly, using peptide mass fingerprinting we show that only a few of the H+-ATPase isoforms are expressed in the Arabidopsis leaf plasma membrane. In present and future work, we are using genetically modified plant lines, to further elucidate specificity and function of 14-3-3 and H+-ATPase isoforms.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

THE ROLE OF DEK1, CR4 AND SAL1 IN CEREAL AND ARABIDOPSIS DEVELOPMENT

Lene Olsen1), Kjetil Fosnes1), Ragnhild Nestestog2), Roy Brown3), Betty Lemmon3), Hilde-Gunn Opsahl-Sorteberg1), Odd-Arne Olsen4) and Stein Erik Lid1)

1) University of Life Sciences, Dept. of Plant and Environmental Sciences, P.O.Box 5003 N-1432 Ås Norway 2) University of Life Sciences, Norwegian Arabidopsis Research Center (NARC), P.O.Box 5003 N-1432 Ås Norway 3) Department of biology, University of Louisiana, Lafayette, LA 70504-2451, USA 4) Pioneer Hi-Bred International, a DuPont Company, Johnston, Iowa 50131- 1004, USA

The endosperm of cereals represents a major source of food, feed and industrial materials, with considerable nutritional and commercial value. The aleurone cell layer containing the majority of oil, high quality proteins, minerals and vitamins as well as the starchy endosperm containing the starch and storage proteins are the most important cell types. Due to the relatively simple structure of the endosperm, it represents an excellent system to dissect molecular and cellular mechanisms in plant development. A better understanding of endosperm development will also allow development of strategies to improve the utilization of this natural resource. Dek1 (Lid et al., 2002), Sal1 (Shen et al., 2003) and Cr4 (Becraft et al., 1996) represent key regulatory genes during cell fate specification and differentiation of aleurone cells. Recent studies of these genes using Arabidopsis as a model plant in both our and other groups, has allowed deeper insight into the mechanisms of gene function. These results as well as ongoing strategies for further functional dissection of gene function are presented.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

DEVELOPMENT OF A SCANDINAVIAN WINTER OAT BY MOLE- CULAR BREEDING AND TRANSGENIC TECHNIQUES Marcus Bräutigam, Aakash Chawade, Gokarna Gharti-Chhetri1, Shakhira Zakhrabekova1, Angelica Lindlöf, Björn Olsson and Olof Olsson2

1Svalöf Weibull AB, Landskrona, Sweden, [email protected] 2Göteborg University, Department of Cell and Molecular Biology, Box 462, SE 405 30 Göteborg, Sweden, [email protected]

Oat is a very promising functional food plant with characters like antioxidants, betaglucans , high fatty acid and protein content. However, before any further improvement of these characters can take place, yield has to be increased. The single most important limitation to oat yield in Sweden is the climate. A Swedish winter oat, which presently does not exist, would increase yield by about 30%. In a previous EST sequencing program we have identified a UniGene set with 2800 sequences from a cold acclimated English winter oat variety denoted Gerald. Among these genes we found 398 sequences with homologies to cold and/or drought-induced genes previously identified in rice, wheat, rye, barley and Arabidopsis, and 51 of these genes encoded putative transcription factors. To further analyse the global gene expression profile in oat during cold acclimation we have developed two different oat microarray platforms, one with 2000 amplified EST clones and one oligo chip that carry 168 genes. In a field trails during two consecutive seasons we have tested about 200 different oat cultivars, selected among the best winter hardy oats available in the world. One of these lines, denoted Pen#65, survived both winters, one of which was severe. Our goal now is to identify key regulated genes important for the winter survival in oat, especially in Pen#65. During spring 2005 we performed several controlled cold acclimation experiments in climate chambers at different time and temperature intervals. Leaf samples from three cultivars, Birgitta, Gerald and Pen#65 all with different cold hardiness were collected and total RNA was then extracted, and global expression studies are now in progress by using the oat microarray platform developed by us. While microarray gene expression data holds tremendous promise, it is too limiting to use such data in isolation. Methods for inferring genetic regulatory networks should therefore combine several types of information. One approach is to combine gene expression data with data on promoter motif over-abundance. Therefore, we have developed a rule-based approach to infer genetic regulatory networks by integrating the information from a combined analysis of promoter motif, gene expression profile relationships, and functional annotation. The model has been tested on previously published microarray data collected from Arabidopsis plants during cold acclimation. This model will know be used on oat micro array data together with extensive promoter analysis in oat. Key regulator genes identified with this approach will be used to develop new molecular markers for an efficient selection of cold tolerant oat varieties in segregating breeding populations. Some of these genes will also be introduced into to commercial oat cultivares by genetic transformation. Work is in progress to establish an Agrobacterium mediated genetic transformation protocol for several oat cultivars.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

DETERMINANTS OF CHILLING TOLERANCE IN ARABIDOPSIS: HOW WE CAN APPLY OUR KNOWLEDGE TO THE SELECTION OF LANDSCAPE PLANTS FOR NORDIC REGIONS

John Einset1; Erik Nielsen2;Erin L. Connolly3; Atle Bones4; Torfinn Sparstad4; Per Winge4 and Jian-Kang Zhu5.

1Norwegian University of Life Sciences, Aas, Norway 1432, 2Donald Danforth Plant Science Center, St Louis MO 63132, 3University of South Carolina, Columbia, SC 29208, 4Norwegian University of Science and Technology, Trondheim, Norway 7491, 5University of California-Riverside, Riverside, CA 92507

Everyone who has examined the problem is surprised that Nordic countries are so restricted in the variety of landscape plants that can be used. This situation becomes even more difficult to understand when one considers the fact that areas in the Northern USA such as Wisconsin and Minnesota which have much colder winters than most of Scandanavia use several types of landscape plants that can not survive in Nordic areas. Over the years, several explanations have been proposed to account for the severity of Nordic environments for plant survival. A demonstrated process involves the fact that imported landscape plants are often poorly adapted in terms of their daylength requirements for hardening in the fall. Without sufficient hardening, plants are overly sensitive to early frost during the fall. On the other hand, there are probably several other processes that are harmful for plants in Nordic regions. This presentation will focus on the idea that chilling contributes significantly to plant stress in Scandanavia, especially during the critical periods of transition during fall and spring when cool days are accompanied by high light intensities. These conditions lead to imbalances in photosynthesis with a strong potential for the generation of reactive oxygen species (ROS), leading to oxidative stress. We have used Arabidopsis as a model to identify new determinants involved in chilling stress. Based on earlier research, we knew that application of glycine betaine (GB) to plants prior to chilling can improve tolerance to chilling stress. We hypothesized that at least part of GB’s effect could be ascribed to the activation of the expression of stress tolerance genes. Using a strategy based on high-throughput gene expression analysis with microarrays and Northerns, we identified 24 genes activated by GB in leaves and 20 genes activated in roots. Several of these GB-activated genes reinforce intracellular processes protecting cells from oxidative damage while others appear to be involved in setting up a scavenging system for reactive oxygen species (ROS) in cell walls. Studies with knockout mutants for two genes activated by GB (the membrane trafficking RabA4c gene and a gene for a putative bZIP transcription factor) demonstrated that the mutants respond only slightly to GB, if at all, compared to wild type in a recovery-from-chilling assay. The knockouts also had higher levels of superoxide in leaves under chilling conditions, especially in the veins of leaves where the expression of the RabA4c gene is localized. Taken together, these results point toward links between oxidative stress, gene expression, membrane trafficking events as well as intra- and extracellular ROS scavenging in relation to GB effects on stress tolerance in plants.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

THE EVOLUTION OF REPRODUCTIVE ORGAN DEVELOPMENT IN SEED PLANTS

Groth, Erika and Engström, Peter

Department of Physiological Botany, Evolutionary Biology Centre, Uppsala University, Villavägen 6, SE-752 36 Uppsala, Sweden.

Seed plants have dominated our planet since the era of the supercontinent Pangea. The first fossils of flowering plants are only about 140 million years old, but the gymnosperms have been around for more than twice that time. The first fossil flowers had a structure similar to modern flowers. We still do not know how the flower originated.

We study the function of transcription factors called MADS-box genes in reproductive organ development in conifers and angiosperms. This work is based on the hypothesis that the evolution of the reproductive organs is mainly driven by changes in the regulatory mechanisms controlling the development of the organs. We are comparing the functions of ortholog MADS-box genes in Norway spruce (Picea abies) and Arabidopsis thaliana from an evolutionary perspective. We are also studying the function of MADS-box genes in the reproductive development of different conifers, in order to better understand the reproductive development of conifers. The aim of our research is to understand the evolution of the mechanisms controlling reproductive organ development in seed plants, and to understand the evolution of these organs.

Norway spruce is one of the most important trees in the forest industry in Sweden. The wood is used as sawn timber, as well as to produce pulp and paper. Despite the importance of conifers to the national economy in many countries, not much is known about the molecular mechanisms that control their development. This is important, because the long generation time of these trees have prevented conifer breeding.

We have in Norway spruce identified a gene, DAL1, believed to be involved in mediating the transition from juvenile to adult phase, and a gene, DAL10, believed to be involved in establishing the reproductive identity of the shoots that will give rise to seed cones and pollen cones. We have created transgenic Norway spruces ectopically expressing these genes, in order to produce trees with a much reduced generation time. This would enable functional genetics in Norway spruce. In the future it might also make conifer breeding possible for the forest industry.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

DETECTION OF EXPLOSIVES USING PLANTS

Carsten Meier

Aresa Biodetection, Sølvgade 14, DK-1307 Copenhagen K, Denmark

Aresa Biodetection (Aresa) is a spin-off from the Institute of Molecular Biology at the University of Copenhagen. Aresa is owned by the DTU Innovation A/S and the investment and development company Bracifeae A/S, which is partly owned by Vækstfonden.

The company has developed and intellectually protected a plant based biodetection system enabling plants to change colour from green to red when growing near by a specific outer stimulus. During the last couple of years Aresa has genetically engineered a large number of plants lines of Arabidopsis thaliana, which are able to change colour when exposed to different types of explosives present in the soil (i.e. landmines, unexploded ordnance (UXO)). The key component responsible for the red colour formation is nitrogen dioxide (NO2), which is a chemical entity formed by reduction of explosive molecules such as TNT, RDX, etc. The break down of explosives is partly mediated by conglomerates of soil bacteria. Metabolites such as NO2 are released during this process. Hence the ability of such bacteria to decompose explosives, and thus using such compounds as energy sources, will serve as a clear advantage for the plant based detection system, when designed to be triggered by NO2.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

INTERACTING PATHWAYS IN ARABIDOPSIS DEFENSE SIGNALING

Palva, E. Tapio; Li, Jing; Kariola, Tarja; Helenius, Elina; Brader, Günter

Viikki Biocenter, Dept of Biological and Environmental Sciences, Div of Genetics, University of Helsinki, POB 56, FI- 00014, Helsinki, Finland

Induced defenses play a major role in plant disease resistance and are controlled by an interconnected signal network with ethylene (ET), jasmonic acid (JA) and salicylic acid (SA) as crucial mediators. Cross-talk between signal transduction pathways is a central feature of this network and potential synergism or antagonism between defense pathways is determined by recognition of a particular type of pathogen or other environmental cues such as light. Our recent data suggest also presence of cross-talk between signal pathways controlling plant defense and abiotic stress tolerance. We have identified WRKY70 as one node of convergence in plant defense signaling integrating signals from the mutually antagonistic SA- and JA-mediated pathways. We employed expression profiling using transgenic plants as well as a WRKY70 knock-out mutants of Arabidopsis to identify the suites of defense-related genes controlled by WRKY70 and showed that WRKY70 overexpression promotes expression of SA-induced genes while suppressing those responsive to JA. This WRKY70-controlled suppression of JA-responsive genes appears to involve NPR1. Modulation of the balance between JA and SA-mediated signaling causes opposite effects on plant resistance to fungal pathogens Alternaria brassicicola and Erysiphe cichoracearum. While upregulation of WRKY70 enhances plant susceptibility to A. brassicicola it causes increased resistance to E. cichoracearum and the bacterial pathogens Erwinia carotovora and Pseudomonas syringae.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

SYNTAXINS SUPPRESS SALICYLIC ACID-MEDIATED DEFENCE

Ziguo Zhang, Jing-long Qiu, Carsten Petersen, Mari-Anne Newman, Angela Feechan, Helge Tippmann & Hans Thordal-Christensen

Dept. of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark

SYP121 (PEN1), identified and described by our laboratory, is an Arabidopsis plasma membrane tSNARE syntaxin protein. SYP121 is directly involved in exocytosis leading to the papilla formation. Its null mutants have dramatically reduced penetration resistance to the barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh), a non-host pathogen on Arabidopsis. An insertion mutation in the closest related gene SYP122 has no effect in penetration resistance. A mutant knocked-out in both SYP121 and SYP122 is necrotic and dwarfs, indicating a shared function of these genes unrelated to penetration resistance. A further characterization of the syp121 mutant showed that SYP121 has a role in the salicylic acid (SA)-mediated defence response. This was studied by introducing mutations in the SA-pathway genes, EDS1, SID2, EDS5 and NPR1, and by introducing the transgene NahG, encoding a SA hydrolase converting SA to catechol. These results tell us that SYP121 acts as negative regulators of the SA-mediated defence response. Taking together, this syntaxin separates penetration resistance and SA-mediated resistance genetically.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

SCREENING FOR SUPPRESSORS OF ARABIDOPSIS ACD11

Frederikke Gro Malinovsky, Peter Brodersen, Daniel Hofius, Jan Joensen, Lea V. McKinney, John Mundy Nikolaj H. T Petersen, Morten Petersen

Institute of Molecular Biology and Physiology; University of Copenhagen

Despite attempts to link plant cell death to animal apoptosis, comparative analyses of the genetic determinants of programmed cell death (PCD) in plants and animals have yet to identify conserved gene functions. Loss of function of the Arabidopsis gene Accelerated Cell Death 11 (ACD11; Brodersen et al. 2002 Genes & Develop. 16, 490; Brodersen et al. 2005 Plant Physiol. 138, 1037) activates vegetative cell death and disease resistance responses dependent upon the hormone salicylate (SA). D Double mutant analysis showed that of 12 mutants affected in defense-associated PCD (eds1, pad4, eds5, sid2, npr1, rar1, pbs1, pbs3, ein2, etr1, jar1, ndr1), only two (eds1 and pad4) suppress acd11 PCD in the presence of the SA analog BTH. To further understand ACD11 function, we are searching for PCD regulators in a large-scale acd11 suppressor screen. More specifically, acd11 homozygotes expressing the bacterial SA hydroxylase nahG were mutagenized and plants surviving BTH treatment isolated as putative suppressors. This identified a number of recessive and dominant suppressors of acd11. Recent work has sorted the recessive mutants fall into fifteen complementation groups. Sequencing has confirmed that two of these groups are allelic to eds1 validating the utility of the screen.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

DEFENCE MECHANISMS IN ARABIDOPSIS TO LEPTOSPHAERIA MACULANS

Christina.Dixelius, Jens Staal, Maria Kaliff

Department of Plant Biology and Forest Genetics, SLU, Box 7080, 750 07 Uppsala Sweden

The hemibiotroph ascomycete Leptosphaeria maculans is the causal agent of blackleg or stem canker on Brassica crops. Our group has focused on a L. maculans – Arabidopsis pathosystem in order enhance our understanding of resistance to this economically important pathogen in a hope to apply our knowledge on any Brassica crop in the future. During mapping attempts of a number of L. maculans susceptible (lms) mutants, a loss-of-resistance phenotype was observed in Col x Ler and Ler x Ws F2 progenies in a 15:1 segregation pattern. Further studies of this loss-of-resistance using Col-4 x Ler-0 and Ler-2 x Cvi-1 recombinant inbred lines (RILs) enabled us to establish a well defined genetic position for the locus that is responsible for resistance in the accessions Col, Ws and Cvi (RLM1Col/Ws/Cvi) on chromosome 1. The RIL screening also located the resistance locus responsible for resistance in the Ler accessions (RLM2Ler) to chromosome 4. Assessments of T-DNA mutants within the genetic position for RLM1 identified two TIR- NB-LRR genes as responsible of RLM1 activity and the most severe mutant was confirmed to be allelic to susceptible Col-0 x Ler-0 in F1 and F2 progenies. The region spanning the locus of RLM1 has an ancient duplication event to chromosome 4 within the region of RLM2, which makes it likely that RLM2 is a paralog of RLM1. In contrast to all previously known TIR-NB-LRR resistance systems, RLM1 and RLM2 are both independent of the EDS1 and PAD4 proteins. Mutations in the R gene signalling components RAR1 and HSP90, but not SGT1b, does however abolish RLM1/RLM2 activity. In addition, a weak co-dominant resistance trait of Col parental origin was identified as a quantitative resistance factor. Screenings of Arabidopsis accessions revealed that all 168 accessions tested, except An-1 (Antwerpen-1), displayed a high degree of L. maculans resistance. We have through microarray-based case-control bulk segregant comparisons of transcriptomes in pools of Col-0 x An-1 progenies identified the absence of a gene (RLM3) that causes susceptibility in An-1. In addition to L. maculans, loss of RLM3 was found to have a negative influence on Alternaria brassicae, A. brassicicola and Botrytis cinerea resistance. The use of T-DNA insertion mutants in Col-0 confirmed the natural deletion found in An-1 to be responsible for the susceptible phenotypes and defined RLM3 activity to a short splice form of the gene. The analysis implies that a short TIR-NB or TIR-X alternative transcript of this gene is responsible for resistance against various pathogens, possibly acting as a down-stream signaling component. Additional defense response factors of importance in this pathosystem will be discussed.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

BARLEY GENOMICS AND PLANT DEFENCE RESPONSES

Collinge, David B.1, Gjetting, Sisse1, Gjetting, Torben 2, Gregersen, Per L. 1,3, Jensen, Michael Krogh 1, Lyngkjær, Michael 2 and Rung, Jesper H. 1

1Department of Plant Biology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark http://www.uk.plbio.kvl.dk/~dacoj3/ [email protected] 2Risoe National Laboratory, 4000 Roskilde, Denmark 3Danish Institute of Agricultural Sciences, Flakkebjerg, Denmark.

The powdery mildew fungus Blumeria graminis f.sp. hordei represents an excellent model for studying defence responses in an intact plant inoculated with a fungus at the molecular level for several good reasons: genetics, cytology and synchronised development of the pathogen, as well as agricultural relevance. The fungus is limited to the epidermal tissues of the host but invokes an extensive defence response in the mesophyll tissues which primarily comprise PR-proteins exhibiting antimicrobial activities against this fungus. We have chosen to apply post-genomic approaches to study transcripts which represent proteins from the epidermal tissues which would appear to have a regulatory role in the defence response. These include the 14-3-3 proteins, NAC-domain protein family of transcription factors and receptor-like protein kinases (RLK). The public barley EST data bases (URL) now contain approx 400,000 barley cDNA sequences, many of which have been obtained from libraries prepared from plants infected with the powdery mildew fungus. Some 300 cDNAs representing 14 different NAC-transcript families have been identified in this resource but rather fewer RLK’s. We have concentrated on two specific RLK’s from barley where there is up to a 40 fold increase in transcript levels in inoculated epidermal tissues of barley. We will present data illustrating the expression of these genes, approaches towards protein–protein interactions involving these genes and RNAi interference experiments to determine their roles in the defence response. We have also obtained Arabidopsis T-DNA insertion lines for several NAC genes and will determine whether they exhibit altered phenotypes with respect to pathogen infection.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

ISOLATION AND CHARACTERIZATION OF PROMOTERS FOR GENES UP-REGULATED IN POWDERY MILDEW INFECTED PLANT CELLS

Pernille Olsen, Torben Gjetting, Peter Hagedorn, Karen L. Olesen and Michael F. Lyngkjær

Biosystems Department, Risø National Laboratory, PO Box 49, 4000 Roskilde, Denmark

Powdery mildew attack of susceptible barley result in a mixture of infected and resistant epidermal cells. Even in the compatible interaction, attacked barley epidermal cells tries to prevent fungal penetration by reinforcing their cell wall. However, this defense is only partially efficient and a number of fungal penetration attempts will succeed and the attacked barley epidermal cell will be infected. Individual gene transcript profiles of non-attacked, infected and resistant barley epidermal cells were obtained by single-cell micro-extraction of mRNA followed by a 10 K cDNA micro array analysis. In order to investigate barley genes involved in the response to powdery mildew attack, promoter fragments for some of the most significant up-regulated genes in infected cells were cloned. Additionally, in silico analyses were used to predict orthologous Arabidopsis genes coding for e.g. a GCN5-related N-acetyltransferase (GNAT), a fungal elicitor immediate early responsive protein (FIERG2), hexose transporters and actin and the promoters for these genes were isolated as well. The activities of the isolated barley and Arabidopsis promoters are currently tested in Arabidopsis plants using gfp and uidA as marker genes. Transgenic T1 Arabidopsis plants are inoculated with the powdery mildew Erysiphe cichoracearum (Eca) to investigate candidate promoters stimulated by powdery mildew infection. To further investigate the function of the candidate genes up- regulated in mildew infected cells, Eca infected Arabidopsis mutant lines will be screened both for changes in infection rates and for changes in the developmental stages of the fungus.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

DEFENSE REACTIONS IN NORWAY SPRUCE TOWARD THE PATHOGENIC ROOT-ROT CAUSING FUNGUS HETEROBASIDION ANNOSUM

Carl Gunnar Fossdal, Ari Hietala, Harald Kvaalen and Halvor Solheim

Norwegian Forest Research Institute, Hogskoleveien 8, 1432 Aas, Norway [email protected]

The root-rot causing fungus Heterobasidion annosumcan attack both spruce and pine trees and is the economically most damaging pathogen in northern European forestry. We have monitored the Heterobasidion annosum S-type (fairly recently named H. Parviporum) colonization rate and expression of host chitinases and other host transcripts in Norway spruce material with differing resistances. Transcript levels of three chitinases, representing classes I, II and IV, were monitored with real-time PCR. We have also transferred a Class IV chitinase to Arabidopsis as well as its promotor in GFP and YFP reporter constructs. Ramets of two 33 -year-old clones differing in resistance were employed as host material and inoculation and wounding was performed. Multiplex real-time PCR detection of host and pathogen DNA was also performed to follow the colonization of the host tissues by the pathogen and the collapse in host DNA levels in infected regions. Host defense transcript levels, as an indicator of the host defense response, were monitored with singleplex real-time PCR. Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. Fourteen days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for the strong clone (589), but had progressed further into the host tissue in the weak clone (409). Transcript levels of the class II and IV chitinases increased following wounding or inoculation, while the transcript level of the class I chitinase declined following these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in 589 than in similar sites in 409 three days after inoculation, suggesting that the clones differ in the rate of pathogen perception and host defense signal transduction. This an earlier experiments using mature spruce clones as substrate indicate that it is the speed of the host response and not maximum amplitude of the host response that is the most crucial component in an efficient defense in Norway spruce toward pathogenic fungi such as H. annosum.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

LIPO-OLIGOSACCHARIDE OF XANTHOMONAS CAMPESTRIS TRIGGERS INNATE IMMUNE RESPONSE IN ARABIDOPSIS THALIANA

Mari-Anne Newman¶, Antonio Molinaro*, Gitte Erbs¶, Alba Silipo*, Luisa Sturiale‡, J. Maxwell Dow§, Rosa Lanzetta*, Michelangelo Parrilli*

¶ Royal Veterinary and Agricultural University (KVL), Frederiksberg, Denmark.*, Università di Napoli, Italy. ‡Istituto per la Chimica e la Tecnologia dei Materiali Polimerici - ICTMP – CNR, Catania, Italy. §BIOMERIT Research Centre, Cork, Ireland. [email protected]

Lipopolysaccharides (LPSs) and lipooligosaccharides (LOSs) are major and vital components of the cell surface of Gram-negative bacteria that have diverse roles in bacterial pathogenesis of animals and plants. In plants, they may contribute to the exclusion of plant-derived antimicrobial compounds, hence promoting the ability of a bacterial pathogen to infect plants. Conversely, LPSs and LOSs can be recognized by plants to elicit or potentiate plant defense- related responses and cause prevention of the hypersensitive response (HR) induced by avirulent bacteria. Almost nothing is known of the molecular basis of the recognition processes that trigger these plant responses. Here we address this issue through the structural determination of the LOS of Xanthomonas campestris pv. campestris strain 8004 and examine the effects of this molecule and fragments obtained from it by chemical treatments on the prevention of HR and the induction of the defense-related gene PR1 and PR2 in Arabidopsis thaliana. LOS and the lipid A and core oligosaccharides derived from it were all able to prevent the HR in Arabidopsis thaliana caused by avirulent bacteria and to induce PR1 and PR2 transcript accumulation. However although LOS induced PR1 and PR2 transcript accumulation in two temporal phases, the core oligosaccharide induced only the earlier phase and lipid A only the later phase. These findings indicate that plant cells can recognize lipid A and core oligosaccharide structures within LPS to trigger defensive cellular responses possibly via different receptors.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

CHARACTERISATION OF STRESS-RELATED MAP KINASE SUBSTRATES IN ARABIDOPSIS

Andreasson E, Merkouropoulos G, Jenkins T, Brodersen P, Thorgrimsen S, Petersen NH, Zhu S, Qiu JL, Micheelsen P, Rocher A, Petersen M, Newman MA, Bjorn Nielsen H, Mattsson O, Peck S, Mundy J

Lund University, Dept of Cell and Organism Biology, Sölvegatan 35, SE-223 62 Lund; Institute of Molecular Biology, University of Copenhagen Oester Farimagsgade 2A, Copenhagen; The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK

Phosphorylation is probably one of the most common post translational modifications, and plays a role in all aspects of cell biology. However, the discovery of identification of substrates for protein serine/threonine kinases has become a rate-limiting step in advancing our knowledge of cell signalling. Often mitogen-activated protein kinase (MAPK) pathways convert extracellular signals to cellular responses including changes in gene expression. Arabidopsis MAP kinase 4 (MPK4) negatively regulates systemic acquired resistance (SAR), and by 2-hybrid screening a component of this pathway called MKS1 (MPK4 substrate 1) was found. MPK4 phosphorylates MKS1, and MKS1 interacts with WRKY transcription factors. Plants that over-express MKS1 exhibit constitutive SAR. A 2-D gel-based phosphoproteomic screen has identified two substrates of AtMPK3 and AtMPK6. Plants that over-express these substrates are resistant to salt and drought stress.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

ON THE ROLE OF EXCESS EXCITATION ENERGY IN REGULATION OF PLANT DEFENCE RESPONSES; HOLISTIC ANALYSIS OF PLANTS’ STRESS SIGNALLING NETWORK

Stanislaw Karpinski

Department of Botany, Stockholm University, 106 91 Stockholm, Sweden Umeå Plant Science Centre, Umeå University, 901 85 Umeå, Sweden

In their natural environment plants are incessantly exposed to abiotic and biotic hazards that may result in increased levels of reactive oxygen species. These species pose a risk but they are also used in induction of defense and acclimatory responses. In our attempts to characterize systemic acquired acclimation we found that this process is associated with active form of cell death similar to that observed in hypersensitive disease defense responses. Our studies have shown that the excess excitation energy (EEE)-induced cell death is dependent on ethylene signaling mediated through EIN2. Ethylene signaling however is not required for acclimation to EEE. By using the lesion simulating disease1 as a model system for loss of light acclimation we have identified that EEE-induced cell death is controlled by LSD1, EDS1, EIN2 and PAD4, which regulate cellular ethylene/ROS/auxin homeostasis. Furthermore, we show that the chloroplasts redox status promotes EEE-dependent cell death. These finds have important implications for understanding of plants’ light acclimatory and defence holistic strategies. Short motifs of many cis-regulatory-elements (CREs) can be found in the promoters of most Arabidopsis genes, and this raises the question of how their presence can confer specific regulation. We developed a new algorithm to test the biological significance of CREs by first identifying every Arabidopsis gene with a CRE and then statistically correlating the presence or absence of the element with gene expression profile on multiple DNA microarrays. This algorithm was successfully demonstrated on previously characterized abscisic acid, ethylene, sucrose and drought CREs in Arabidopsis, showing that the presence of these elements indeed correlates with treatment-specific gene induction. Later, we used standard motif-sampling methods to identify 128 putative excess light, reactive oxygen species, and sucrose induced motifs. Our algorithm was able to filter 20 out of 128 novel CREs which significantly correlated with gene induction by either heat, reactive oxygen species and/or sucrose. The position, orientation, and sequence specificity of CREs was tested in silicio by analyzing the expression of genes with naturally occurring sequence variations. In three novel CREs, the forward orientation correlated with sucrose induction and reverse orientation with sucrose suppression. Functionality of some novel CREs was experimentally confirmed using Arabidopsis cell suspension cultures transformed with short promoter fragments or artificial promoters fused with reporter gene. Our genome-wide analysis opens up new possibilities for in silicio verification for the biological significance of newly discovered CREs, and allows for subsequent selection of such CREs for experimental studies.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

DIURNAL CHANGES IN MYROSINASE-GLUCOSINOLATE SYSTEM: EFFECTS OF PHOTOPERIOD AND TEMPERATURE

De Felice, Annavera; Jørstad, Tommy; Rohloff, Jens and Bones, Atle Magnar

Cell and Molecular Biology Group, Department of Biology, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway

Glucosinolates, sulphur, and nitrogen containing secondary metabolites, together with the enzyme myrosinase (β-thioglucosidase) constitute a well- know defence system that characterizes the plants belonging to the order Capparales (Rodman et al., 1998). This order also includes the model plant Arabidopsis thaliana and other agriculturally important plants such as oilseed rape and Brassica vegetables. Glucosinolates themselves are not toxic but upon tissue damage, they are hydrolysed by myrosinase yielding isothiocyanates, thiocyanates, and nitriles that mediate the response to the attack. Although constitutively present, myrosinase and glucosinolates represent a dynamic system, able to react quickly to defence inducing stimuli (Pontoppidan et al. 2005; Mewis et al., 2005) by varying in enzyme activity levels and compound composition and content (Koritsas et al., 1991; Bodnaryk, 1992).The dynamicity of the system is not restricted to defence reactions but has also been demonstrated in plants not subjected to any pest. In fact, there is evidence of a high rate of glucosinolate catabolism in intact plants, Brown et al. (2003) showed that glucosinolate levels decrease in A. thaliana germinating seeds few days after planting. Furthermore, Rosa et al. (1998) showed that glucosinolate levels in B. oleracea intact plants, undergo dramatic changes during 24 hours under the influence of the photoperiod and also of the temperature and that these changes follow ultradian rhythms. Since there are only few studies conducted in physiological conditions the objective of our study was to gain more knowledge about the glucosinolate metabolism and myrosinase activity regulation in intact plants. We analysed in parallel glucosinolate levels and myrosinase activity in rosette leaves of A. thaliana wild type plants during 24 hours cycles. The mean glucosinolate contents were not affected by day/night changes, but they varied significantly during the 24 hours showing oscillatory tendencies. In the same way, myrosinase activity seemed not to be modulated by photoperiod but to follow cyclic oscillations during the 24 hour periods. The effect of temperature on glucosinolate levels and myrosinase activity diurnal changes was also investtigated. The exposurure to 10 degrees higher temperature for 24 hours did not signify- cantly affect neither the myrosinase activity nor the total glucosinolate content in A. t. leaves, while differences were found for individual glucosinolates.

Bodnaryk, R.P., Phytochemistry, 31, 2671-2677 (1992). Brown, P.D., Tokuhisa, J.G., Reichelt, M., Gershenzon, J., Phytochemistry, 62, 471-481 (2003). Koritsas, V.M., Lewis, J.A., Fenwick, G.R., Ann. Appl. Biol., 118, 209-221 (1991). Mewis, I., Appel, H.M., Hom, A., Raina, R., Schultz, J.C., Plant physiology, 138: 1149-1162 (2005). Pontoppidan, B., Hopkins, R., Rask, L., Meijer, J., Plant Science, 168: 715-722 (2005). Rodman, J.E., Soltis, P.S., Soltis, D.E., Sytsma, K.J., Karol, K.G., Am. J. Bot., 85: 997-1006 (1998). Rosa, E.A.S., Rodriguez, P.M.F.,J. Sci. Food Agric., 78, 208-212 (1998).

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

ERD15 MODULATES DISEASE RESISTANCE AND FREEZING TOLERANCE IN ARABIDOPSIS

Kariola, Tarja; Brader, Günter; Helenius, Elina; Li, Jing; Heino, Pekka and Palva E. Tapio

Department of Biological and Environmental Sciences, Genetics, University of Helsinki, P.O.B 56, FIN-00014, Helsinki, Finland

ERD15 (early responsive to dehydration) is rapidly induced in response to various biotic and abiotic stress stimuli in Arabidopsis. Transgenic plants overexpressing ERD15 showed improved resistance to the bacterial necrotroph Erwinia carotovora and this was accompanied by enhanced induction of SAR- marker genes PR1 and PR2. In response to stress the transgenic plants accumulated more abscisic acid (ABA), but simultaneously the sensitivity of these plants to this phytohormone was decreased, which was seen for example as reduced tolerance to drought*. It seems that insensitivity to ABA improves the resistance of Arabidopsis to E. carotovora. This is supported by the fact that the tolerance of the abscisic acid insensitive 1 and 2 (abi1 and abi2) mutants for E. carotovora infection is also enhanced compared to wild-type plants. Recent studies point towards post-transcriptional mRNA processing as a way of influencing the expression of ABA-regulated genes. ERD15 has been shown to have a PAM2-motif through which it binds to poly(A)-binding protein (PABP), an important component of translational machinery in eukaryotes. We propose that ERD15 is a novel mediator of ABA-signalling in both biotic and abiotic* stress responses of Arabidopsis and that this signalling is mediated through post-transcriptional regulation.

* See the accompanying poster of Elina Helenius, University of Helsinki

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

POSTERS

ABSTRACTS (alphabetical order)

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

THE ROLE OF NITRIC OXIDE IN O3-INDUCED CELL DEATH

Kollist, Hannes1; Ahlfors, Reetta1; Brosché, Mikael1; Radhika, Desikan2 and Kangasjärvi, Jaakko1

1Plant Biology, Department of Bio- and Environmental sciences, University of Helsinki, P.O Box 65 (Viikinkaari 1), FIN-00014, Finland 2Centre for Research in Plant Science, Genomics Research Institute, University of the West of England, Bristol BS16 1QY, United Kingdom

Nitric oxide (NO) has been shown to be involved, together with reactive oxygen species (ROS), in activation of various stress responses in plants but biochemical mechanisms by which ROS and NO participate in these reactions are still unclear. Also, ozone (O3) is known to elicit plant stress responses and changes in hormonal signaling eventually leading to cell death in sensitive species. Accordingly, it is likely that also NO is involved in the activation of O3-induced cell death reactions. However, the role of NO in the O3-induced cell death still remains to be elucidated. In this study two O3-sensitive mutants rcd1 and rcd3 and two knockout alleles of Arabidopsis nitric oxide synthase (AtNOS) were chosen in order to study the role of NO in O3-induced cell death. To this point we have used confocal microscopy to analyze O3-induced NO emissions. These studies revealed that, indeed, O3 induces rapid accumulation of NO. Emission started from the guard cells and eventually moved to epidermis and mesophyll. In addition, we have found clear differences between the studied mutants. For example, initial O3-induced NO burst is delayed in AtNOS and rcd3. Furthermore, experiments were plants were either treated with NO or were NO was removed also supported the importance of NO in O3- induced cell death. These results will be discussed in respect to the latest data obtained.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

VIRUS-INDUCED GENE SILENCING IN ARABIDOPSIS THALIANA

Albrechtsen, Merete

Biotechnology Group, Department of Genetics and Biotechnology, Danish Institute of Agricultural Sciences Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Phone (+45) 35 28 25 82, E-mail [email protected]

Virus-induced gene silencing (VIGS) is a powerful tool for creating functional gene knockouts in plants. The technique is faster than stable transformation, even in Arabidopsis thaliana. Furthermore, genes that are essential for plant growth and development can be studied using VIGS, and several genes can be targeted at once. Several viral vectors have been reported to be useful for VIGS in A. thaliana. We are currently testing and comparing three viral vectors based on either Tobacco rattle virus or Cabbage Leaf Curl Virus, for their ability to silence the endogenous phytoene desaturase gene in A. thaliana. In addition we have modified two of the vectors for more easy inoculation by agroinfiltration.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

GENETIC DETERMINANTS OF FLOWER SYMMETRY IN GERBERA HYBRIDA

Broholm, Suvi; Laitinen, Roosa; Teeri, Teemu H. and Elomaa, Paula

Department of Applied Biology, University of Helsinki, P.O. Box 27, FIN-00014 Helsinki, Finland

Gerbera hybrida (Asteraceae) is an interesting model plant for floral symmetry studies as the Gerbera inflorescence is formed of morphologically different types of flowers. The three flower types (ray, trans and disc) differ in their size, symmetry and sexuality and are densely packed so that the inflorescence resembles a single radially symmetrical flower. In marginal ray flowers, three of the five petals are fused together forming a large corolla and causing a bilaterally symmetric shape of the flowers. Towards the centre of the inflorescence, the flowers gradually become smaller and more symmetrical until the centremost disc flower is radially symmetric. Only disc flowers are hermaphrodite with both male and female sexual identity whereas ray and trans flowers have only female sexual identity.

The traditional model plant for floral symmetry research has been Antirrhinum majus. Analysis of the flower symmetry mutants in Antirrhinum has led to the identification of four genes (CYC, DICH, RAD and DIV) that encode proteins controlling the dorsoventral asymmetry of Antirrhinum flowers. CYC and DICH promote dorsal identity and encode DNA-binding proteins that belong to the TCP family, which most probably are modulators of cell-division-related gene expression. RAD and DIV genes encode proteins of the large family of MYB transcription factors and are needed to identify dorsal and ventral petal identity, respectively. So far, two CYC-like and two DIV-like genes have been isolated from Gerbera. My aim is to investigate the molecular basis of floral symmetry in Gerbera by characterising the function of these candidate genes by expressional analysis and studying genetically modified plants.

The morphological differences between Gerbera ray and disc flowers occur early in their development. We have used quantitative real time PCR to compare the expression of the four candidate genes separately between the Gerbera ray and disc flowers in early developmental stages. The PCR results showed that of these candidate genes, GhCYC2 is more strongly expressed in ray flowers than in disc flowers. The CYC gene in Antirrhinum is known to regulate the asymmetry of petals and the arrest of stamen development. These results suggest that GhCYC2 gene might take part in the differentiation of the Gerbera ray flowers. To study this further, we are making in situ hybridisation analysis and transgenic Gerbera lines.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

METABOLITE PROFILE OF CHLOROPLASTIC GLUTATHIONE PEROXIDASES (GPXS) IN ARABIDOPSIS THALIANA PROVIDES A NOVEL INSIGHT BETWEEN PHOTO-OXIDATIVE STRESS AND BASAL PATHOGEN RESISTANCE MECHANISMS

Christine Chi-Chen Chang1, Thomas Moritz2, Krister Lundgren2, Hans Stenlund2, Michael Melzer3, Philip M. Mullineaux4, Barbara Karpinska1, Stanislaw Karpinski1,*

1Department of Botany, Stockholm University, Frescati, SE-106 91, Stockholm, Sweden, 2Dept. Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, SE-901 87, Umeå, Sweden, 3Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany, 4Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom.

Glutathione peroxidases (GPXs, EC: 1.11.1.9) are important reactive oxygen species (ROS) scavengers because of their broader substrate specificities and stronger affinity for H2O2 than catalases (CATs). The main function of GPXs are believed to catalyze the reduction of hydrogen peroxides, organic hydroperoxides and lipid hydroperoxides to water or alcohol respectively by using reduced glutathione (GSH) as the electron donor. We have previously shown that transgenic lines (AS-cpGPX) with reduced expression of genes encoding chloroplastic isoforms of glutathione peroxidases (GPXs, EC: 1.11.1.9) were more sensitive to photo-oxidative stress as compared to control plants. Here, we show that reduced chloroplastic GPX activity is associated with increased size of intercellular spaces in foliar spongy mesophyll, higher foliar hydrogen peroxide levels, higher accumulation of starch in chloroplasts and higher foliar anthocyanin content. Metabolite profile analysis revealed that GPX7 deficient mutant and transgenic As-cpGPX lines with the strongest reduction of cpGPX activity were the most divergent among all analysed lines and when compared to control plants cultivated under either high light or low light conditions. Presented results show that cpGPX activity, photo-oxidative stress tolerance, chloroplastic reactive oxygen species metabolism and resistance mechanisms to virulent bacteria are functionally linked in plants.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

VESICLE TRAFFICKING AND ABIOTIC STRESS

Angela Feechan, Zigou Zhang, Jin-long Qiu, Helge Tippmann, Carsten Pedersen and Hans Thordal-Christensen

Dept. of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen, Denmark

SNAREs drive secretory vesicle targeting and fusion. Recently syntaxins from the SNARE (Soluble N-ethylmaleide-sensitive factor (NSF) adaptor protein) superfamily have been implicated in plant adaptation to both abiotic and biotic stress responses (Pratelli et al., 2004). The Arabidopsis Syntaxin of Plants121 (AtSYP121) was previously shown to be required for non-host resistance to barley powdery mildew (Collins et al., 2003). NtSyr1, which is the tobacco homolog of AtSYP121, was shown to block abscisic acid (ABA) mediated Cl- and K+ channel responses in guard cells, implicating a role for NtSyr in drought responses (Leyman et al., 1999). Therefore we have been investigating the role of the plasma membrane SYP121 and the closely related syntaxin SYP122 in abiotic stress responses. We have found that syp121 and syp121syp122 double mutant seedlings show increased sensitivity to ABA. The single mutants syp121, syp122 and the double mutant syp121syp122 show divergent responses during heat shock and salt stress compared to wild type Col-0 seedlings. We are currently studying the molecular basis of these altered responses.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

ERD15 MODULATES ABIOTIC STRESS RESPONSES IN ARABIDOPSIS

Kariola, Tarja; Helenius, Elina; Brader, Günter; Li, Jing; Heino, Pekka and Palva, E. Tapio

Dept of Biological and Environmental Sciences, Genetics, University of Helsinki, P.O.Box 56, 00014 University of Helsinki, Finland

ERD15 (early responsive to dehydration) is rapidly induced in response to various abiotic and biotic stress stimuli in Arabidopsis. We have studied the effect of either overexpression or RNAi silencing of ERD15 on abiotic stress tolerance in Arabidopsis*. RNAi silencing of ERD15 improved the freezing tolerance of non-acclimated plants. After acclimation at +4°C, both the overexpression and RNAi lines were able to cold acclimate. However, the lines overexpressing ERD15 were impaired in their ability to increase their freezing tolerance after treatment with the phytohormone abscisic acid (ABA). The silencing of ERD15 also improved the drought tolerance of the transgenic plants, as well as made the seeds of these plants hypersensitive to ABA in germination. Taken together, the effects seen in the plants with modulated ERD15 expression are considered to be due to altered responsiveness to ABA. This indicates that ERD15 affects abiotic stress responses through ABA.

*See also the abstract of Tarja Kariola, University of Helsinki.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

IDENTIFICATION OF CONSTANS-LIKE 3 (COL3) AS A POSITIVE REGULATOR OF LIGHT SIGNALING AND ROOT GROWTH.

Sourav Datta1, G. H. C. M. Hettiarachchi1, Xing-Wang Deng2 and Magnus Holm1

1) CMB-Molecular Biology, Gothenburg University, Medicinaregatan 9C, Box 462, 405 30 Gothenburg, Sweden. 2) Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Conecticut 06520-8014

COP1 is an E3 ubiquitin ligase that represses photomorphogenesis in the dark. Proteins interacting with COP1 could therefore be important regulators of light dependent development. Here we identify COL3 as a novel interaction partner of COP1. A GFP-COL3 fusion protein co-localizes with COP1 to nuclear speckles when transiently expressed in plant cells. This localization requires the B-box domains in COL3, indicating a novel function of this domain. A loss-of- function col3 mutant has longer hypocotyls in red light and in short days. Unlike co, the col3 mutant flowers early and shows a reduced number of lateral branches in short days. The mutant also exhibits reduced formation of lateral roots. The col3 mutation partially suppresses the cop1 and det1 mutations in the dark, suggesting that COL3 acts downstream of both these repressors. However, the col3 mutation exerts opposing effects on cop1 and det1 in terms of lateral roots and anthocyanin accumulation, suggesting that COL3 also has activities that are COP1 and DET1 independent. In conclusion, we have identified COL3 as a positive regulator of photomorphogenesis that acts downstream of COP1 but can promote lateral root development independently of COP1 and also function as a day length sensitive regulator of shoot branching.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

REGULATION OF THE DEG2 SERINE PROTEASE

Huesgen, Pitter; Schuhmann, Holger and Adamska, Iwona

Department of Biology, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany

The Deg/HtrA proteases are ATP-independent serine endopeptidases which are present in most organisms, including bacteria, humans and plants. Previous work in our laboratory has shown that the Deg2 protease of the model plant Arabidopsis thaliana selectively degrades the photodamaged D1 protein in the reaction center of photosystem II (PSII) in vitro. Therefore, Deg2 is thought to catalyze the primary cleavage of photodamaged D1 protein, which is an important step of the repair mechanism that restores functional PSII. Our present studies aim to elucidate the regulation of the Deg2 protease activity, especially with regard to its D1 degrading activity. We found Deg2 associated to the stromal side of the thylakoid membranes and as a soluble protein in the chloroplast stroma. The amount and distribution of Deg2 protein remained unchanged after exposure to different light intensities, which suggest either a substrate regulation or a posttranslational regulation of the D1 degrading activity of Deg2. Recent advances on Deg2 regulation and complex formation will be presented.

Keywords: proteolysis; chloroplast; DegP/HtrA; D1 turnover

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

ARE CALCIUM PUMPS INVOLVED IN SIGNAL TRANSDUCTION IN GUARD CELLS?

Jakobsen, Mia Kyed; Schiøtt, Morten*; Bækgaard, Lone*; Webb, Alex and Palmgren, Michael G*.

Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK. *Department of Plant Biology, KVL, Thorvaldsensvej 401, 1871 Frederiksberg, DK.

Guard cells are responsive to a wide array of environmental and endogenous signals including, light, temperature, oxidative stress, abscisic acid, gibberellins, fungal elicitors and nodulation factors. Transduction of the signals have been shown to occur through oscillations in cytosolic free calcium1. We are interested in how these oscillations are generated. Entry of calcium into the cytosol occurs through the opening of calcium permeable channels, which allow downhill flow of calcium ions from intra- or extracellular stores such as the vacuole and the cell wall2. Removal of calcium from the cytosol is thought to involve Ca2+-ATPases and H+/Ca2+-exchangers, which use ATP or electrochemical gradients to pump calcium ions out of the cytosol. To elucidate whether calcium pumps are involved in signal transduction in guard cells, we have generated T-DNA insertion mutants and double mutants of two calcium pumps, ACA8 and ACA10, belonging to the P-type ATPase super family of ion transporters in Arabidopsis. In addition, we are generating a mutant overexpressing a hyper-activated N terminal-deleted version of ACA8. ACA8 localizes to the plasma membrane3 and both ACA8 and ACA10 are highly expressed in guard cells. We use infrared themography and stomatal aperture assays to characterize mutant guard cell responses to various environmental cues. The calcium reporter cameleon is employed to study cytosolic calcium oscillations by fluorescence ratio imaging.

1Allen et al. (2000). Alteration of stimulus-specific guard cell calcium oscillations and stomatal closure in Arabidopsis det3 mutant. Science 289: 2338-2342. 2Peiter et al. (2005). The vacuolar Ca2+-activated channel TPC1 regulates germination and stomatal movement. Nature 434: 404-408. 3Bonza et al. (2000). At-ACA8 encodes a plasma membrane-localized calcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus. Plant Physiol 123: 1495- 1505.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

THE FUNCTION OF RCD1 PROTEIN AND ITS ROLE IN PLANT STRESS SIGNALING

Jaspers, Pinja; Kuusela, Tiina and Kangasjärvi, Jaakko

Plant Biology, Department of Biological and Environmental Sciences, University of Helsinki, PO Box 65 (Viikinkaari 1), FIN-00014 Helsinki, Finland

RCD1 (radical-induced cell death1) is an Arabidopsis thaliana protein whose function is essential in regulating reactive oxygen species-related signaling. The gene was originally identified through an ozone sensitive mutant rcd1 that is not only sensitive to increased levels of ozone and superoxide but also has several alterations in its hormonal signaling.

There is no known biochemical function for RCD1 protein but it is thought to be localized to the nucleus and to have two domains involved in protein-protein interactions (WWE domain and a ”C-terminal domain”). Two earlier studies have revealed several interaction partners for RCD1. In addition, RCD1 contains the catalytic core of ADP-ribosyl transferases (ADPRT) and the protein sequence contains many potential post-translational modification sites. Available DNA microarray data suggests that the protein is more likely to be regulated post-translationally than on the RNA level. The Arabidopsis genome contains a close homolog of RCD1 called SRO1 and the characterizatoin of these two proteins will be conducted in parallel.

This study contains four main parts: i) the characterization of the protein-protein interactions of RCD1 by repeating the yeast-2-hybrid studies with a ”stress cDNA library” and by fishing interactors from plants with a tagged RCD1 protein ii) the analysis of the biochemical activity and the targets of RCD1 by using pure heterologously expressed protein iii) the study of RCD1 activity regulation by potential post-translational modifications iv) the localization of the protein by GUS and GFP constructs as well as in situ hybridizations

The information gained by the biochemical characterization of RCD1 will be combined with systemic biology approaches to gain insights to the regulation and transmission of plant stress signaling.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

TOWARDS MAPPING OF GENE(S) CONTROLLING RESISTANCE IN CABBAGE (BRASSICA OLERACEA) TO BLACK ROT (XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS)

Jensen, Brita Dahla,d; Joshi, Sharadab; Bimb, Haric; Holm, Henrik Lehmannd; Torp, Anna Mariad; Andersen, Sven Boded aDepartment of Plant Biology, Plant Pathology Section, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark; bPlant Pathology Division and cBiotechnology Unit, Nepal Agricultural Research Council, Khumaltar, Lalitpur, Nepal; and dDepartment of Agricultural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark

Black rot, caused by the bacterium Xanthomonas campestris pv. campestris (Xcc) is considered one of the most serious diseases of crucifers world-wide, for instance in low elevation tropics. We have demonstrated that the breeding line ‘Badger I-16’, released for plant breeding programmes worldwide exhibited a high level of disease resistance under field conditions in Tanzania to a widespread, endemic common race of the pathogen (race 1). The existence of pathogenic variants (races) in Xcc creates concern regarding the specificity and durability of resistance sources. However, Badger also exhibited resistance to another common race, race 4, under field conditions in Nepal. This indicates that ‘Badger’ exhibits field resistance of a race nonspecific nature. Few sources of resistance in B. oleracea to common races are known, making ‘Badger’ an extremely interesting resistance source for further study as well as for practical utilization. Previously, it has been reported that the resistance in ‘Badger’ was controlled by one major gene, and that the expression in the heterozygous condition was influenced by one recessive and one dominant modifier gene. ‘Badger’ exhibited a higher level of resistance to Xcc than any other cultivar in the field in Tanzania and Nepal, indicating that the resistance in ‘Badger’ has not been fully exploited in breeding programmes, presumably due to the complex genetic background and inheritance. An F2 population (240 plants) based on a cross between ”Badger” (resistant) and ”Copenhagen Market Biro” (susceptible) has been screened for disease resistance to Xcc, race 4, and was found to segregate for disease resistance in a normally distributed manner, suggesting that quantitative trait loci are underlying the resistance. Currently, we are in the process of mapping the resistance trait on the basis of the F2 population. So far, 5 microsatellite (SSR) and 13 AFLP markers have been used to construct a focused map with 3 linkage groups that have revealed 4 QTLs associated with the resistance trait. In three of the mapped genes the favourable alleles are derived from ‘Badger’. We will now saturate the linkage groups with more SSR markers to anchor and orientate them in comparison with previously published B. oleracea maps. In the future we wish to use ‘comparative’ mapping and ‘candidate genes’ from Arabidopsis to saturate the map further and to reveal the mechanisms involved in the resistance.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

FROM YELLOW TO GREEN BY RESTORING THE CHLOROPHYLL SUPPLY

Andreas Hansson and Poul Erik Jensen

The Royal Veterinary and Agricultural University, Department of Plant Biology, Plant Biochemistry Laboratory, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark.

A SALK line (chl27) carrying a T-DNA insert in the promoter region of CHL27, the Arabidopsis homologue to Chlamydomonas Crd1, has a chlorotic phenotype. Disrupting the promoter region results in reduced expression of CHL27. Upon feeding with delta-aminolevulinic acid in darkness, chl27 accumulate Mg- protoporphyrin IX monomethyl ester, indicating that CHL27 is required for protochlorophyllide synthesis and makes CHL27 a candidate for one of the subunits of the aerobic cyclase. Biochemical and genetical characterisations has previously shown that at least two or three protein components are required for aerobic cyclase activity.

Compared with wild-type the amount of chlorophyll in chl27 plants is reduced by 70% and the chlorophyll a/b ratio is increased twofold. Electron micrographs of the chloroplast ultrastructure revealed that chl27 has a twofold increase in the proportion of stroma lamellae and 40% decrease in grana membranes. Blue- native PAGE and western blot analysis demonstrated an almost complete loss of LHCII protein and a significant reduction of PSI in chl27. When the chl27 cDNA controlled by a 35S promoter is introduced in to the chl27 plant, the plant recover and get indistinguishable from wild-type regarding chlorophyll level, chlorophyll a/b ratio and 77K fluorescence. Our aim is to construct a plant with an inducible expression of CHL27 and to use the recovery phase to study the assembly of pigment-protein complexes.

Attempts to identify the other protein components of the aerobic cyclase have been made. After sucrose-gradient centrifugation of WT thylakoids, CHL27 is found in the same fractions as LHCII. When thylakoids form a chlorophyll b deficient plant is loaded on the gradient, CHL27 is still found in the same fractions as for WT, however now in the absence of LHCII. The other proteins found in these fractions remain to be characterised.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

WAVELENGTH DEPENDENCE OF EXPRESSION OF UV-B-INDUCED MOLECULAR MARKERS IN ARABIDOPSIS THALIANA.

Kalbina, Irinaa, Li, Shaoshana,b,Björn, Lars Olofc, Strid,Åkea,* aInstitutionen för naturvetenskap och Centrum för livsvetenskap, Örebro universitet, SE-70182 Örebro, Sweden; bSchool of Life Science, South China Normal University, Guangzhou 510631, China; cInstitutionen för cell- och organismbiologi, Lunds universitet, Sölvegatan 35, SE-22362 Lund, Sweden.

Action spectroscopy is an important method for the understanding of the impact of solar UV-B radiation. An action spectrum describes the relative effectiveness of quanta of different wavelengths in producing a particular biological response. The biological response could, for instance, be effects at the molecular level, such as DNA damage, or at the organism level, such as growth rate. The main reason for determining action spectra is to identify specific chromophores involved in light perception leading to a corresponding response. Fluence-response curves and action spectra were obtained for mRNA transcripts of four UV-B molecular markers: CHS (encoding chalcone synthase), PYROA (encoding an enzyme involved in formation of pyridoxine), MEB5.2 (encoding a protein with unknown function which is strongly up-regulated by UV-B), and LHCB3 (encoding a chlorophyll a/b binding protein) in Arabidopsis thaliana in the range of 280-360 nm. The obtained results suggest the existence of two distinct UV-B photoreceptors: one sensitive around 300 nm and the other sensitive around 280-290 nm. Among the investigated molecular markers, CHS and PYROA expression was regulated through the chromophore absorbing around around 300 nm, whereas MEB5.2 and LHCB3 expression was regulated through the chromophore absorbing at 280-290 nm. A minor peak at 350 nm in the action spectra of MEB5.2 and possibly CHS also suggests a response in the UV-A region due to absorption by a UV-A absorbing chromophore. Possible candidates for the different chromophores, are discussed. The results obtained show that a network of UV-B signal transduction pathways exists that regulates the expression of the four molecular marker genes.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

CANDIDATE GENE GLABROUS1 AND ADAPTIVE VARIATION IN TRICHOME PRODUCTION IN ARABIDOPSIS LYRATA

Kivimäki, Maarit; Kärkkäinen Katri, Gaudeul Myriam, Løe Geir and Ågren Jon

Finnish Forest Research Institute, P. O. Box 18, FIN-01301 Vantaa, Finland [email protected]

Adaptation to local environment is an important feature in evolutionary biology, but the genetic basis of adaptation is still poorly known. It is yet unclear whether adaptive traits are controlled by several genes with small effect or only a few genes with large effect – and if the important genes have a regulatory function or code for structural proteins. Model species with good genetic knowledge integrated with field studies of natural populations will be useful to connect adaptive phenotypic variation and DNA-level variation. We have used trichome production in the natural populations of Arabidopsis lyrata as a simple model to study the genetic basis of adaptive variation. In leaf hair (trichome) production, there is polymorphism and both glabrous and trichome-producing morphs exist. Trichomes protect the plant for example against herbivores but may be associated with a cost. Field studies conducted in four polymorphic and one monomorphic (glabrous) population in the Swedish A. lyrata showed trichomes to reduce herbivory. Next, candidate gene(s) were chosen on the basis of genetic studies in the plant model species A. thalaina and we characterized variation in GLABROUS1 in the A. lyrata populations. We also analysed microsatellite variation in the same populations. Three interesting mutations in the third exon of the regulatory gene GLABROUS1 were discovered. This sequence variation was found to be strongly associated with phenotypic variation whereas the neutral genetic markers showed no hidden population structure causing the association, suggesting that selection is acting on the gene. Thus, we conclude that GLABROUS1, a transcription factor of the Myb-gene family, explains natural adaptive variation in the species A. lyrata.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

UNCOVERING PEST RESISTANCE SUSCEPTIBILITY DIFFERENCES BETWEEN TWO ARABIDOPSIS THALIANA ECOTYPES THROUGH INFESTATION WITH M. PERSICAE AND B.BRASSICAE.

Kuśnierczyk, Anna; Winge, Per; Midelfart, Herman; Bones, Atle

Cell and molecular biology group (CMBG), Department of Biology, The Norwegian University of Science and Technology, Realfagbygget, 7491Trondheim, e.mail: [email protected]

The phloem piercing-sucking insects are an important factor in limiting crop plants production. With the steady growth of the world population and the food shortage, there is a rising interest to enhance the plant endogenous resistance to insect pests. One of the key points on the way to success is to understand the plant molecular response to phloem feeding. Using Arabidopsis thaliana and aphids as a model system of plant-insect interaction, recent studies attempt to investigate the diversity of plant resistance mechanisms on gene expression level. Although aphids developed a feeding strategy, which minimizes cell damage, there is some evidence that infestation activates broadly similar defence pathways as pathogen attack, chewing herbivory and wounding. Little is known about distinct changes in gene expression profiles upon infestation with different species of phloem feeders. Also variation in gene induction between the diverse ecotypes of the same plant has not been yet elucidated. We use microarray gene expression analysis to investigate transcriptional profiles of two A.thaliana ecotypes infested with two species of aphids. The Arabidopsis thaliana ecotypes Cape Verde Islands (Cvi) and Wassilewskija (Ws) were infested with a generalist pest - Myzus persicae (green peach aphid), and a specialist - feeding exclusively on brassicaceous hosts - Brevicoryne brassicae (cabbage aphid). Additionally growth rates of the infested and aphid free plants were measured to evaluate the general effect on plant growth. Our work shows that the genes involved in JA-signalling pathway, glutathione associated genes and calcium-dependent signalling proteins are the main responders in plants infested for 72 hours. The higher induction of some defensive proteins in Cvi ecotype, especially AOS and LOX – two of the key enzymes in JA- pathway, points towards Cvi being more resistant than Ws ecotype. In addition, our studies of changes in the growth rate confirm this thesis. Comparison of the response profiles to generalist and specialist aphid also reveals some significant differences. The largest dissimilarities in expression are found in PR-1, putative copper amine oxidase and myrosinase associated proteins.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

FUNCTION OF THE RCD1-SRO GENE FAMILY: A SYSTEMS BIOLOGY APPROACH

Kuusela, Tiina1; Sipari, Nina2; Jaspers, Pinja1; Keinänen, Markku2 and Kangasjärvi, Jaakko1

1Plant Biology, Department of Biological and Environmental Sciences, University of Helsinki, PO Box 65 (Viikinkaari 1), FI-00014 Helsinki, Finland 2 Department of Biology, University of Joensuu, PO Box 111, (Yliopistokatu 7), FI-80101 Joensuu, Finland

High-throughput techniques for monitoring RNA and protein levels are on their way to becoming mainstream tools for utilizing the vast amount of genetic information available. Combined with metabolic profiling, transcriptomics and proteomics allow the detection of the gene function on a systems biology level and the identification of processes involved in plant development and stress responses. The rcd1 (radical-induced cell death1) mutant of Arabidopsis has been shown to be defective in the containment of programmed cell death and in the signaling of several plant hormones. RCD1 belongs to a novel gene family with 5 unknown genes encoding proteins distinctively similar to RCD1 (SRO1- SRO5; SIMILAR TO RCD-ONE 1-5). Interestingly, a conserved domain of ADP-ribosylation has been assigned to all the RCD1-SRO proteins. The systems biology study of the RCD1-SRO protein function includes characterisation of T-DNA insertion lines (i. e. “knock-out”-lines) with large- scale (24K, MWG-Biotech) microarray analysis, metabolite profiling as well as proteomics. Metabolite profiling has been carried out with a new LC-ESI/MS method for non-targeted, comprehensive analysis of the small molecular weight metabolites. Also specific groups of compounds relevant to stress responses and ADP-ribosylation, for instance nucleotides, nucleosides and lipids, are chosen for targeted analysis. The biological importance of the RCD1-SRO proteins is discussed in respect to the latest results obtained from microarrays and metabolic studies.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

KNOCKING OUT CHLOROPLASTS

Per Mühlenbock and Stanislaw Karpinski

Department of Botany, Stockholm University, Lilla Frescativägen 5, 106 91, Stockholm, Sweden

Chloroplasts and peroxisomes are the major contributors of reactive oxygen species (ROS) during the photoperiod (Asada 1999, Foyer and Noctor 2003) An increasing amount of evidence is pointing to an important role of chloroplasts for the regulation the defence signaling in plants. (Chaerle 2004, Mateo 2004, Samuilov 2000, Seo 2000). In order to discern the chloroplast derived signals from signals that originate from other cellular compartments we evaluate several different strategies of knocking out chloroplasts in vivo. The strategies involve physiological and pharmaceutical treatments and analysis of the applicability of variegated mutants of Arabidopsis thaliana.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

LOCALIZING PLASMA MEMBRANE PROTON ATPASE EXPRESSION IN ARABIDOPSIS

Markus Müller1, Magnus Alsterfjord2, Marianne Sommarin1,2

1Umeå University, Department of Plant Physiology, 90187 Umeå 2Lund University, Department of Plant Biochemistry, P.O. Box 124, 221 00 Lund

Plasma membrane localized proton ATPases are a well characterized family of singlesubunit proton translocators in plants. They establish an electrochemical gradient across the plasma membrane which is used as driving force for numerous transport processes. This includes nutrient as well as water (stomata opening). The regulation of these proteins involves the phosphorylation and subsequent binding of 14-3-3 proteins to the autoinhibitory C-terminus. However, little is known about the isoform specific functions of the 11 genes present in Arabidopsis. We started a general approach to localize the expression of most AHA promoters in Arabidopsis and by the same time study the effects of RNAi plants of these genes. All invesigated promoters show distinct localization within the plant, thus indicating tissue-specific functions for each family member. The AHA10 promoter responds strongly to the availability of sucrose and salt stress. In addition, we show that the promoter of AHA2 is inversely regulated by auxin in root and leaf tissue.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

THE NORWEGIAN ARABIDOPSIS RESEARCH CENTER

Ragnhild Nestestog, Stein Erik Lid and Odd Arne Rognli.

University of Life Sciences, Dep. of Plant and Environmental Sciences, P.O BOX 5003, N-1432 Ås, Norway.

A national service and competence centre in plant genomics

The Norwegian Arabidopsis Research centre (NARC) is one of the 10 national technology platforms funded through the Functional Genomics (FUGE) initiative of the Norwegian Research Council. NARC is initially funded for five years (2002-2007), and will offer resources and technology in the Arabidopsis thaliana model system. NARC will establish a solid base for fundamental and applied plant science in Norway. The partners of the centre provide basic technologies for the utilization of functional genomics tools in plant science. The platform offer services such as provision of clones for micro arrays, transformation and protein-protein interactions. The establishment of service centres will result in more efficient use of resources and equipment available in the Norwegian plant science community.

At the University of Life Sciences (UMB) we perform transformation, genotyping, seed multiplication, maintenance of seed collection and a vector bank. The transformation of Arabidopsis allows the detection of specific genes (GUS or GFP) under the control of gene specific, inducible or constiutive promoters. Fusion of the gene of interest to reporter genes or epitope tags allows examination of subcellular localization, and the addition of his-tags also makes it possible to isolate the protein encoded by the specific gene. In addition to gene expression analysis, the phenotypic effects are also studied.

Large T-DNA knockout collections (SALK, GABI-Kat, SAIL, INRA) are available to the scientific community, from which T-DNA insertions in a gene of interest may be identified. Individual plants from specific T-DNA lines are usually genotyped in order to discriminate between plants carrying the insertion in heterozygous or homozygous condition. Currently, genotyping of T-DNA lines is being carried out at NARC.

NARC is built on existing competence in Arabidopsis research present in Norway, and is a collaboration between Arabidopsis researchers at three institutions; Norwegian University of Science and Technology (DNA micro arrays, bioinformatics), University of Oslo (Yeast two hybrid system, In Situ hybridisation) and The University of Life Sciences (transformation, genoytping, seed bank, vector bank). An exclusive collection of Arabidopsis ecotypes from northern latitudes, mutants and GUS marker lines are also available at NARC (UMB) (www.umb.no/narc).

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

FUNCTIONAL CHARACTERISATION OF ABI4 AND TINY-LIKE AP2 TRANSCRIPTION FACTORS IN ARABIDOPSIS THALIANA

Övernäs, Elin; Engström, Peter and Söderman, Eva

Dept Physiological Botany, EBC, Uppsala University, Villavägen 6, S-752 36 Uppsala, Sweden

The AP2 (APETALA2) and EREBPs (ethylene-responsive element binding proteins) are members of a family of transcription factors that is apparently unique to plants. The family comprises 145 members and is characterized by the presence of the AP2 DNA binding domain. On the basis of number of repetitions and sequence of the AP2 domain, the family has been classified into five groups, the APETALA2, RAV, DREB and ERF subfamilies and others. The DREB subfamily contains 56 members, where several proteins have been shown to be involved in environmental and stress responses. Members include ABI4, shown to be involved in regulating seed responses to ABA and/or seed- specific signals, TINY and five proteins that have been shown to be involved in drought and/or cold responses in the plant. Phylogenetic analysis of the deduced amino acid sequences of the DREB subfamily have identified 6 subclasses, A1- A6. We are interested in studying the function of a group of protein encoding genes closely related to ABI4 and TINY, of subclass A4. Based on microarray data from Genevestigator, light and environmental stress conditions regulate the expression of genes in this subgroup. We are studying the function of two closely related genes in the A4 group, AtAP2.5 and AtAP2.8, by analysis of T- DNA insertion mutants and transgenic Arabidopsis plants with high level of expression of the genes. Plants that express AtAP2.5 or AtAP2.8 at high levels have a phenotype similar to the tiny mutant, with a reduction in root and hypocotyl length and small and narrow cotyledons and leaves, indicating a role as negative regulators of cell expansion. AtAP2.5 and AtAP2.8 are both expressed in different organs of the flower, and AtAP2.8 is expressed in germinating seeds and young seedlings. Currently we are investigating the response to ABA, in regards to seed germination and root elongation in the mutants and overexpressor plants.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

DIFFERENTIAL Mn EFFICIENCY IN BARLEY GENOTYPES IS RELATED TO DIFFERENCES IN Mn UPTAKE CAPACITY IN THE LOW nM CONCENTRATION RANGE

Pai Pedas1, Thomas P. Jahn1, Jan K. Schjørring1, Peter E. Holm2 and S. Husted1

1Plant & Soil Science Laboratory, Department of Agricultural Sciences, The Royal Veterinary & Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen 2Department of Natural Sciences, The Royal Veterinary & Agricultural University

There is considerable variability among barley (Hordeum vulgare L.) genotypes in their ability to grow in soils containing low levels of plant available manganese (Mn). The physiological basis for this tolerance to low Mn availability is unknown. In the present work Mn2+ influx in barley roots of the Mn efficient genotype Vanessa and the Mn inefficient genotype Antonia was examined. Two separate Mn transport systems, mediating high-affinity Mn2+ influx between 0.1 -130 nM and low-affinity Mn2+ influx between 10 nM to 2 mM Mn, respectively, were identified in both genotypes. The two genotypes differed only in high-affinity kinetics with the Mn efficient genotype having almost four times higher Vmax than the inefficient genotype, but similar Km values. This difference was further documented in long-term Mn uptake experiments where plants were exposed to chemostatic low nM Mn concentrations. It is concluded that differential capacity for high-affinity Mn influx contributes to differences between barley genotypes in Mn-efficiency.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

LOCALISATION OF LIGHT STRESS PROTEINS IN PHOTOSYNTHETIC COMPLEXES OF ARABIDOPSIS THALIANA

Verena Reiser and Iwona Adamska

Universität Konstanz; Lehrstuhl für Pflanzenbiochemie und –Physiologie; Fachbereich Biologie; Universität Konstanz; 78457 Konstanz

Eukaryotic photosynthetic organisms and cyanobacteria respond to light stress by transient accumulation of light stress proteins from the Elip (early light induced protein) family. Elips in higher plants are nuclear-encoded proteins located in the thylakoid membranes and related to the chlorophyll a/b-binding proteins of photosystem I and II. The Elip-family consists of three types of proteins: Elips (early light-induced proteins), Seps (stress-enhanced proteins) and Ohps (one-helix proteins). It is assumed that Elip-family members participate in binding of free chlorophyll molecules released during the degradation of pigment – protein complexes under stress conditions. Thereby they might prevent the formation of free radicals and/or are acting as sinks for excess excitation energy. Sucrose density centrifugation of solubilized, native light-stressed thylakoids followed by gel filtration enabled us to isolate one single Elip 1-containing complex. In this complex the presence of light- harvesting proteins (LhcpII) could be verified with western blots, showing an interaction between Elip 1 and the Lhcp II. Further analyses of the complex are in progress.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

HETEROTRIMERIC G-PROTEINS POSSIBLE ROLE IN UV-B- STRESS SIGNALLING IN ARABIDOPSIS THALIANA

Ristilä, Mikael; Kalbina, Irina and Strid, Åke

Department of Natural Sciences, Örebro University, SE-701 82 Örebro, Sweden

Decreasing ozone concentrations in the stratosphere has led to an increase in UV-B-radiation reaching the earth’s surface. Due to its high energy content UV-B radiation has a negative impact on many living organisms, some effects seen in plants are: DNA damage and damage to the photosynthesis apparatus. When exposed to UV-B radiation plants induce different defence mechanisms. Many parts of the UV-B induced signalling pathways are unknown, including the perception of the radiation by the plant.

Heterotrimeric G-proteins consists of three subunits, α, β and γ. Arabidopsis thaliana has only one gene encoding the α-subunit and one for the β-subunit, but two different γ-subunit genes. Recent studies has shown that heterotrimeric G-proteins are involved in many different types of signalling pathways, such as cell proliferation, stomatal responses and phytohormone signalling. The aim of our study was to determine whether heterotrimeric G-proteins also participate in the signal transduction of UV-B-stress.

We have performed gene expression and phenotype studies on knockout plants of Arabidopsis thaliana exposed to UV-B-stress. We have studied knockouts of the α-subunit (gpa1-1, gpa1-2), the β-subunit (agb1-1), and the agb1-2 gpa1-4 double mutant. We have also done studies on plants overexpressing the β- subunit.

The stress marker genes we have studied were CHS; PYROA; MEB5.2; PR-5 and LHCB1.6. These are meant to represent different UV-B-signalling pathways. In the phenotype studies the UV-B-induced reduction in biomass was studied, comparisons between the different mutants and wild type were made.

We could not detect any differences in the expression patterns of the studied genes when comparing the different G-protein mutants with wild type plants. As for the phenotype experiments, the β-subunit mutant (agb1-1) seem to be less sensitive to UV-B-stress compared with wild type, whereas the two gpa- mutants seem to be as sensitive towards UV-B-stress as wild type.

We conclude that heterotrimeric G-proteins do not play a significant role in the signalling of UV-B-stress. Although, we cannot rule out that they might be involved in some process, as our phenotype experiments show.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

GLUCOSINOLATE METABOLISM AND DEGRADATION IN ARABIDOPSIS THALIANA IS AFFECTED BY LIGHT?

Rohloff, Jens; De Felice, Annavera and Bones, Atle Magnar

Cell and Molecular Biology Group (CMBG), Department of Biology, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway

Secondary metabolites from plants comprise a vast diversity of chemical structures showing distinct functions related to plant growth and reproduction, signalling, protection and stress response. Although many of these compounds are involved in basic physiological processes, their biosynthesis, turnover and genetical regulation with regard to abiotic growth factors were less understood until recently due to functional genomic analyses. Diurnal rhythms and oscillation patterns of concentration levels of secondary metabolites such as flavonoids, alkaloids and terpenes have been reported. Arabidopsis thaliana is known for its content of glucosinolates, which are also found in other species of the Brassicaceae family. Degradation of glucosinolates by the plant’s own enzyme myrosinase upon tissue damage leads to the release of biologically active and toxic volatiles of mainly isothiocyanates and nitriles, thus underscoring their major function in plant defence. Glucosinolates are also supposed to be involved in auxin regulation and sulphur assimilation, but only few reports describe their functionality related to diurnal rhythms and light parameters. Based on studies carried out at the Department of Biochemistry (Prof. Gershenzon) at the Max Planck Institute for Chemical Ecology at Jena in Germany, we describe results from Arabidopsis studies focusing on glucosinolate degradation levels and patterns upon day-night shift and differing light intensities. Analyte detection was carried by headspace solid-phase microextraction (SPME) (Rohloff & Bones, 2005) and/or solvent extraction (SE) (modified after Lambrix et al., 2001), coupled with gas chromatography and mass spectrometry (GC-MS). Cyclic fluctuations of isothiocyanates from autolysed samples could be revealed by SPME application in three independent day-night cycles. These results are confirmed by simultaneous SE sampling at distinct time-points from the same experiments. In contrast, short (1 h) and long-time adaption (3 d) to different light intensities did not lead to obvious differences in glucosinolate degradation patterns, although no-light or low light (125 µmol/s/m2) conditions seemed to favour higher glucosinolate levels.

The presented results underscore the hypothesis of a continuous biosynthesis and turn-over of glucosinolates in Arabidopsis thaliana, being further investigated in on-going studies with KO mutants by means of genomic, proteomic and metabolic analyses.

References Lambrix, V., Reichelt, M., Mitchel-Olds, T., Kliebenstein, D.J. & Gershenzon, J. 2001. The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory. The Plant Cell 13: 2793-2807. Rohloff, J. & Bones, A.M. 2005. Volatile profiling of Arabidopsis thaliana – Putative olfactory compounds in plant communication. Phytochemistry 66: 1941-1955.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

NARC – NORWEGIAN ARABIDOPSIS RESEARCH CENTRE – UNIVERSITY OF OSLO

Barbro E. Sæther and Reidunn B. Aalen Norwegian Arabidopsis Research Centre, Dept. of Molecular Biosciences, University of Oslo, P.O box 1041, Blindern, N-0316 Oslo, Norway

NARC is a national technology platform, sponsored by the Norwegian Functional Genomics initiative (FUGE), that will offer Norwegian plant scientists resources and technology in the Arabidopsis thaliana model system. FUGE is the result of an initiative taken by the Norwegian research establishment; the underlying process has been supported by the Research Council of Norway. FUGE represents a cooperative effort between Norway’s universities and research institutions and the industrial sector. NARC will establish a solid basis for basic plant molecular biology, applied plant research and plant breeding in Norway. The centre partners provide the basic technologies to utilize the powerful tools of comparative genomics and thereby to promote the quality of plant science in Norway. The establishment of NARC will result in more efficient use of resources and equipment available in the Norwegian plant science community. By taking advantage of the tools already available, gene function and interaction of gene products can be studied in great detail. NARC is built on existing competence in Arabidopsis research at the Norwegian University of Science and Technology (NTNU), University of Oslo and the Norwegian University of Life Sciences (UMB), and has a close collaboration with other FUGE resource centres, e.g. The Norwegian Micro Array Consortium, imaging and proteomics facilities and bioinformatics expertise centres. At NARC - University of Oslo we focus on In situ hybridisation for cellular localisation and expression analysis of specific transcripts, and Yeast two- hybrid analysis for protein-protein interactions and identification of binding domains and interacting/assoaciated protein partners.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

ARABIDOPSIS AS A HOST FOR PRODUCTION OF ORAL VACCINES

Thulin, Sara1; Scherbak, Nikolai2; Sävenstrand, Helena2; Andersson, Sören1 and Strid, Åke2

1Department of Clinical Microbiology and Immunology, Örebro University Hospital, SE-701 85 Örebro, Sweden 2Department of Natural Sciences, Örebro University, SE-701 82 Örebro, Sweden

Infectious diseases are a global health problem and although success with several vaccination programmes, there is still a lack of protection against a majority of pathogens. Most vaccines produced so far, are injectable and require sterile equipment. This procedure would be much simplified with edible vaccines and with a low production cost, the vaccines could be delivered to a larger part of the population.

Arabidopsis thaliana is used as a first model system and we are focusing on antigens from the sexual tranmissible infections Clamydia trachomatis and HIV. The open reading frame of MOMP protein, the immunodominant antigen from Chlamydia were cloned into pPCV742 and pGreen0029 and transformed into Arabidopsis. In addition the open reading frame of p24, a major core protein of HIV, was also cloned and transformed. Transformants of Arabidopsis of are now beeing analysed and characterized.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

PLANT SAD PROTEINS: CHARACTERIZATION OF TETRAMERIC PISUM SATIVUM PROTEIN– A MEMBER OF THE SHORT-CHAIN REDUCTASE/DEHYDROGENASE FAMILY.

Scherbak, Nikolai; Strid, Åke

Institutionen för naturvetenskap och Livsvetenskapligt centrum, Örebro universitet, S-70182 Örebro, Sweden

In Pisum sativum, the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). The SAD genes are transiently expressed in plants by short exposures to ultraviolet-B radiation, which in turn leads to formation of SAD protein in leaf and stem tissue upon prolonged irradiation.

The recombinant SAD-C protein (which was most highly expressed of the two isoforms) was shown to be a tetramer that probably consists of a dimer of dimers and which possesses quinone-reducing capability. The enzyme shows approximately the same NADH- and NADPH-dependent activity with 2,5- and 2,6-dimethylbenzoquinone and menadione as substrates.

Western blotting shows that the SAD protein is present to a smaller or larger extent in all the different pea tissues examined. The pea seed is the entity where the largest content of SAD is present. However, in the seed, the seed coat shows the lowest amount of enzyme. Environmental stress such as UV-B radiation clearly increases the content of SAD in leaf and stem tissue but not in roots. This indicates that increased expression of the SAD genes, as a result of UV-B exposure, is limited to the exposed tissue (leaves and stem), i.e. a local effect rather than a systemic response (no increased levels in roots). This is substantiated by the heterologous expression from the pea SAD-C promoter in Arabidopsis during wounding. Only the wound site and the vicinal tissue shows transcription from this promoter.

In non-stressed tissue (as well as in UV-B-stressed leaves and stem), SAD occurrance in epidermal or sub-epidermal cells predominates as judged by IHC. Very evident SAD distribution in the protoderm of the pea seed cotyledonary axis is also revealed. These are the most heavily stained cells found in the present study and indicate a possible role for the SAD protein in development as well as in protection against environmental stress. However, expression around vascular tissue is also apparent. Although IHC is not a quantitative method, our results indicate that the UV-B-induced increase of SAD content in pea leaf and stem tissue found by immunoblotting is mainly confined to epidermal and subepidermal cells of the particular surface facing the UV-B radiation source, where the highest intensities of UV-B radiation would penetrate the cells.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

CHROMATIN INSULATORS IN PLANTS

Simola, Carl-Johan and Sundås-Larsson, Annika

Department of Physiological Botany, Evolutionary Biology Center, Uppsala University. Villavägen 6, SE-75236 Uppsala, Sweden.

The aim of my work is to investigate the effects of chromatin insulators on gene regulation in plants.

Chromatin insulators represent a class of regulatory DNA-protein complexes. Chromatin insulators function in two different ways, physical barriers were the insulator forms a barrier and prevents spreading of condensed chromatin, and enhancer blocking were the insulator interfere with enhancer-promotor interactions. Chromatin insulators have been found in a range of organism, e.g.: the scs and scs’ elements in Drosophila, the chicken β-Globulin Locus and the HM mating-type locus in yeast. I will investigate if chromatin insulators are present in plants, and identify potential interacting partners.

Another task is to investigate if there is evolutionary conservation between the plant and mammalian insulator system. This will be done by introducing the key component of the mammalian insulator system, CTCF, in plants and investigate if it give rise to any alterations in development or gene expression.

Detecting an insulator system in plants will reveal new information about gene regulation in plants and could have direct implications in molecular plant breeding.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

INFLORESCENCE DEFICIENT IN ABSCISSION CONTROLS FLORAL ORGAN ABSCISSION IN ARABIDOPSIS.

Grethe-Elisabeth Stenvik1, Melinka A. Butenko1, Sara E. Patterson2, and Reidunn B. Aalen1

1 The Arabidopsis Group, Program for Molecular Genetics, Department of Molecular Biosciences, University of Oslo, Postboks 1041, Blindern, 0316 Oslo. 2 Department of Horticulture, University of Wisconsin-Madison, Madison, Wisconsin 53706-1381.

Abscission is an active process that enables plants to shed unwanted organs. Because the purpose of the flower is to facilitate pollination, it often is abscised after fertilization. We have identified a floral organ abscission mutant in Arabidopsis, inflorescence deficient in abscission (ida). ida is the first floral abscission mutant characterized which shows a complete lack of floral organ abscission. The ida gene encodes a small protein with an N-terminal signal peptide, suggesting that the IDA protein is a ligand of an unknown receptor involved in the developmental control of floral abscission (Butenko et al, 2003 Plant Cell 15: 2296-2307). IDA::GUS reporter lines showed the wild-type IDA gene to be expressed specifically in the floral abscission zone during the time of abscission. A single copy IDA::GUS line has been crossed to the ethylene insensitive mutant etr1-1 to investigate the IDA expression in the etr1-1 background. In addition we have used molecular markers for chitinase and cellulase to track the abscission process in the ida background. Transient GFP expression showed IDA to be localized in the apoplastic space of onion cells. Stable GFP transformants are currently being investigated to look at GFP- expression in planta. In addition we are working on identifying the receptor, and other genes that are components in the same developmental pathway; by doing both yeast two hybrid and activation tagging. Over expression lines will hopefully give us an indication to whether IDA is involved in regulating the last step of the floral abscission, or inhibiting a repair process during abscission.

Hopefully the identification of proteins interacting with IDA will give us further insight into the regulation of the floral abscission process in Arabidopsis.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

EFFECTS OF GLUCOSINOLATES AND THEIR HYDROLYSIS PRODUCTS IN RESISTANCE AGAINST MICROBIAL PATHOGENS

Tran, Diem Hong; De Felice, Annavera; Rohloff, Jens and Bones, Atle Magnar

Cell and Molecular Biology Group (CMBG), Department of Biology, Norwegian University of Science and Technology (NTNU), N-7491 Trondheim, Norway

Glucosinolates (GLSs) a group of defensive metabolites in plants are, upon tissue damage, hydrolysed by myrosinase resulting in formation of biologically active products. The glucosinolate-myrosinase defence system is exclusive for plants of the order Capparales, including the model plant Arabidopsis thaliana. Numerous experiments have indicated GLSs as important defensive compounds though there is relatively little evidence for their role in effective defence against herbivores and pathogens. Other important roles of GLSs are anticarcinogenic and influencing flavour and/or health characteristics of Brassicaceous vegetable, oil and fodder crops. In previous studies, focus has either been on GLSs or hydrolysis products of GLSs. In this study, GLSs and their hydrolysis products will be investigated simultaneously. A profile of GLSs and hydrolysis products and their content in wt and myrosinase T-DNA knockout mutants has been obtained. Most studies on the glucosinolate- myrosinase system have been focusing on plant-insect interaction while little attention has been given to the glucosinolate-myrosinase system’s role in the resistance against microbial pathogens. A model system, Pseudomonas syringae infection on A. thaliana ecotype Columbia-0 is being used in this study to investigate response of glucosinolate-myrosinase system to microbial infections.

In order to elucidate the complex interaction between plants and microbes, metabolic profiles and myrosinase activity were determined for wt and myrosinase T-DNA knockout mutants, both healthy and bacterial infected plants. Preliminary results showed no induction of myrosinase activity upon microbial infection, whereas different ‘constitutive’ levels of myrosinase activity did not seem to influence the level of resistance against microbial pathogens. Correlation between myrosinase activity and glucosinolate content could neither be found, though plants with different levels of myrosinase activity accumulated different types of GLSs upon microbial infection. One important aspect in the development of agricultural crops, breeding for resistance and specific GLS content, is the assumption of a correlation between GLSs and their hydrolysis products. However, the degradation process of GLSs by myrosinase is complex as it is affected by associated proteins/cofactors and reaction conditions. In our study, a correlation between GLSs and their hydrolysis products was found. The present (P. syringae) model is being further investigated in on-going studies combining data from metabolic and transcriptional profiling.

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

IDENTIFICATION OF NEW REGULATORS IN ROS INDUCED CELL DEATH

1Brosché, Mikael; 1Wrzaczek, Michael; 1Kollist, Hannes; 2Kollist, Triin and 1Kangasjärvi, Jaakko

1Plant Biology, Department of Biological and Environmental Sciences, Viikki Biocenter, University of Helsinki, POB 65 (Viikinkaari 1), 00014 Helsinki, Finland. 2Institute of Molecular and Cell biology, University of Tartu, Riia str. 23, Tartu, Estonia

Short and high pulses of the atmospheric pollutant ozone (O3) cause the formation of reactive oxygen species (ROS) in the apoplastic space of leaves. Furthermore, pathogen infection and other biotic and abiotic stresses induce ROS formation. This triggers cell death lesions in the leaves of sensitive plants. ROS trigger the hypersensitive response-like programmed cell death (PCD) that is a common feature of plant responses to ozone and pathogens.[hk1] This PCD appears to be an active process during which ROS exhibit signaling functions in inter- and intracellular communication contrasting earlier views that regarded ROS merely as toxic to cells. While central components to pathogen recognition and signaling have been identified the key elements in ROS perception remain elusive. Receptor-like kinases (RLKs) exhibit important functions in the sensing of pathogens. Subsequently signaling networks are triggered including phosphorylation and de-phosphorylation events that discharge into adjustment of gene expression. Ultimately this leads to a co-ordinated defense response. Taken together it is conceivable that ROS are perceived and processed in a similar way to pathogens, potentially sharing several signaling components.[hk2]

To identify new regulators in the ozone induced cell death response, microarray experiments were performed with ozone exposed wildtype Columbia. About 700 genes show at least a two-fold regulation by the treatment (250 ppb ozone, 6 hours). 60 of these genes were selected for further studies and T-DNA knockouts in these genes were obtained. The knockouts were screened with O3 and sensitive plants were identified. These include amongst others components in calcium signaling, a putative lipase, various proteins of unknown function and two RLKs. This data suggests the involvement of phorphorylation cascades in ROS signaling. Furthermore, both small extracellular proteins as well as RLKs might be directly involved in ROS perception. The study of the biological function and the biochemical properties of the identified proteins will allow for new insights into ROS perception and early ROS signaling. Furthermore these results will help to understand the role of ROS signaling in other biotic and abiotic stresses.[hk3]

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

List of participants

Reetta Ahlfors Sweden Department of Biological and Tel: +46 46 222 36 58 Environmental Sciences E-mail: [email protected] University of Helsinki Viikinkaari 1 Kim Andresen 00014 Helsinki Department of Molecular Biosciences Finland University of Oslo Tel: +358 9 191 59448 P.O. Box 1041 Blindern E-mail: [email protected] N-0316 Oslo Norway Merete Albrechtsen Tel: +47 95070487 Biotechnology Group E-mail: [email protected] Department of Genetics and Biotechnology Danish Institute of Agricultural Sciences Feechan Angela Thorvaldsensvej 40 Department of Agricultural Sciences DK-1871 Frederiksberg C The Royal Veterinary and Agricultural Denmark University Tel: +45 3528 2582 Thorvaldsensvej 40 E-mail: [email protected] DK-1871 Frederiksberg C Denmark Vibeke Alm Tel: +45 3528 3788 Department of Molecular Biosciences E-mail: [email protected] University of Oslo P.O. Box 1041 Blindern Tetsuhiro Asada N-0316 Oslo Institute of Biotechnology Norway University of Helsinki Tel: +47 22 85 45 73 Viikinkaari 1 E-mail: [email protected] 00014 Helsinki Finland Magnus Alsterfjord Tel: +358 9 191 59424 Department of Plant Biochemistry E-mail: [email protected] Lund University Box 124 Kwadwo Owosu Ayeh SE-221 00 Lund Norwegian University of Life Sciences Sweden N-1432 Ås Tel: +46 462229382 Norway E-mail: [email protected] Tel: +47 64 96 56 06 E-mail: [email protected] Ellen Dehnes Andersen Department of Molecular Biosciences Patrick Bienert University of Oslo Department of Agricultural Sciences P.O. Box 1041 Blindern The Royal Veterinary and Agricultural N-0316 Oslo University Norway Thorvaldsensvej 40 Tel: +47 22 85 45 71 DK-1871 Frederiksberg C E-mail: [email protected] Denmark Tel: +45 3528 3419 Erik Andreasson E-mail: [email protected] Department of Cell and Organism Biology Lund University Atle Bones Sölvegatan 35 Department of Biology SE-223 62 Lund Norwegian University of Science and

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

Technology Thorvaldsensvej 40 Høyskoleringen 5 DK-1871 Frederiksberg C N-7034 Trondheim Denmark Norway Tel: +45 3528 3356 Tel: +47 73598692 E-mail: [email protected] E-mail: [email protected] Annavera De Felice Marcus Bräutigam Department of Biology Department of Cell and Molecular Biology Norwegian University of Science and Göteborg University Technology PO Box 462 Høyskoleringen 5 405 30 Göteborg N-7034 Trondheim Sweden Norway Tel: +46 31 773 32 58 Tel: +47-73551280 E-mail: [email protected] E-mail: [email protected]

Suvi Broholm Christina Dixelius Department of Applied Biology Dept of Plant Biology and Forest Genetics University of Helsinki Swedish University of Agricultural Latokartanonkaari 7 Sciences 00014 Helsinki Box 7080 Finland 750 07 Uppsala Tel: +358 9 191 57944 Sweden E-mail: [email protected] Tel: +46-018-673234 E-mail: [email protected] Mikael Brosché Department of Biological and John Einset Environmental Sciences Norwegian University of Life Sciences University of Helsinki N-1432 Ås Viikinkaari 1 Norway 00014 Helsinki Tel: +47 6496 5306 Finland E-mail: [email protected] Tel: +358 19159436 E-mail: [email protected] Carl G. Fossdal Norwegian Forest Research Institute Christine Chi-Chen Chang (Skogforsk) Department of Botany Høgskoleveien 8 Stockholm University N-1432 Ås Lilla Frescativaegen 5 Norway SE-106 91 Stockholm Tel: +47 64949005 Sweden E-mail: [email protected] Tel: +46-8-163770 E-mail: [email protected] Anja Thoe Fuglsang Department of Plant Biology Jan Chojecki The Royal Veterinary and Agricultural PBL University Norwich Research Park Thorvaldsensvej 40 Colney Lane DK-1871 Frederiksberg C Norwich NR4 7UH Denmark UK Tel: +45 3528 3329 Tel: +44 1603 456500 E-mail: [email protected] E-mail: [email protected] Dale Godfrey David B. Collinge Department of Agricultural Sciences Department of Plant Biology The Royal Veterinary and Agricultural The Royal Veterinary and Agricultural University University Thorvaldsensvej 40

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

DK-1871 Frederiksberg C S-405 30 Gothenburg Denmark Sweden Tel: +45 3528 3788 Tel: +46 31 773 3299 E-mail: [email protected] E-mail: [email protected]

Paul Grini Kirsten Bagge Holm Department of Molecular Biosciences Department of Agricultural Sciences University of Oslo The Royal Veterinary and Agricultural P.O. Box 1041 Blindern University N-0316 Oslo Thorvaldsensvej 40 Norway DK-1871 Frederiksberg C Tel: +47 22 85 45 71 Denmark E-mail: [email protected] Tel: +45 3528 3471 E-mail: [email protected] Erika Groth Department of Physiological Botany Anne Honkanen Uppsala University Institute of Biotechnology Villavägen 6 University of Helsinki SE-75236 Uppsala Viikinkari 4 Sweden 00014 Helsinki Tel: +46 18 471 28 07 Finland E-mail: [email protected] Tel: +358 919159425 E-mail: [email protected] Josefine Nymark Heglund Department of Agricultural Sciences Pitter Huesgen The Royal Veterinary and Agricultural Department of Biology University University of Konstanz Thorvaldsensvej 40 Universitätsstrasse 10 DK-1871 Frederiksberg C D-78457 Konstanz Denmark Germany Tel: +45 3528 3465 Tel: +49-7531-882908 E-mail: [email protected] E-mail: [email protected]

Elina Helenius Thomas P. Jahn Department of Biological and Department of Agricultural Sciences Environmental Sciences The Royal Veterinary and Agricultural University of Helsinki University Viikinkaari 5 Thorvaldsensvej 40 00014 Helsinki DK-1871 Frederiksberg C Finland Denmark Tel: +358 9 191 59085 Tel: +45 3528 3484 E-mail: [email protected] E-mail: [email protected]

Chamari Hettiarachchi Mia Kyed Jakobsen CMB - Molecular Biology Department of Plant Sciences Gothenburg University University of Cambridge Medicinaregatan 9C Downing Street S-405 30 Gothenburg CB2 3EA Cambridge Sweden UK Tel: +46 31 773 3297 Tel: +44 1223 766 543 E-mail: E-mail: [email protected] [email protected] Pinja Jaspers Magnus Holm Department of Biological and CMB - Molecular Biology Environmental Sciences Gothenburg University University of Helsinki Medicinaregatan 9C Viikinkaari 1

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

00014 Helsinki Viikinkaari 1 Finland 00014 Helsinki Tel: +358 9 191 59439 Finland E-mail: [email protected] Tel: +358 9 191 59444 E-mail: [email protected] Lilli Sander Jensen Department of Agricultural Sciences Saijaliisa Kangasjärvi The Royal Veterinary and Agricultural Laboratory of Plant Physiology and University Molecular Biology Thorvaldsensvej 40 University of Turku DK-1871 Frederiksberg C Tykistökatu 6A Denmark FI-20520 Turku Tel: +45 3528 37 88 Finland E-mail: [email protected] Tel: +358 2 333 7076 E-mail: [email protected] Brita Dahl Jensen Department of Plant Biology Tarja Kariola The Royal Veterinary and Agricultural Department of Biological and University Environmental Sciences Thorvaldsensvej 40 University of Helsinki DK-1871 Frederiksberg C Viikinkaari 5 Denmark 00014 Helsinki Tel: +45 3528 3439 Finland E-mail: [email protected] Tel: +358 9 191 59092 E-mail: [email protected] Poul Erik Jensen Department of Plant Biology Stanislaw Karpinski The Royal Veterinary and Agricultural Department of Botany University Stockholm University Thorvaldsensvej 40 Lilla Frescativaegen 5 DK-1871 Frederiksberg C SE-106 91 Stockholm Denmark Sweden Tel: +45 3528 3340 Tel: +46-8-161 214 E-mail: [email protected] E-mail: [email protected]

Henrik Johansson Maarit Kivimäki Department of Physiological Botany Finnish Forest Research Institute Uppsala University P. O. Box 18 Villavägen 6 FIN-01301 Vantaa SE-75236 Uppsala Finland Sweden Tel: +358-10 211 2578 Tel: +46 18 471 28 07 E-mail: [email protected] E-mail: [email protected] Hannes Kollist Irina Kalbina Department of Biological and Department of Natural Sciences Environmental Sciences Örebro University University of Helsinki Fakultetsgatan 1 Viikinkaari 1 SE-70182 Örebro 00014 Helsinki Sweden Finland Tel: +46 19301033 Tel: +358 9 191 57788 E-mail: [email protected] E-mail: [email protected]

Jaakko Kangasjärvi Triin Kollist Department of Biological and Department of Biological and Environmental Sciences Environmental Sciences University of Helsinki University of Helsinki

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

Viikinkaari 1 Jakob Markvart 00014 Helsinki Department of Plant Biology Finland The Royal Veterinary and Agricultural Tel: +358 9 191 57788 University E-mail: [email protected] Thorvaldsensvej 40 DK-1871 Frederiksberg C Anna Kusnierczyk Denmark Department of Biology Tel: +45 3528 3340 Norwegian University of Science and E-mail: [email protected] Technology Høyskoleringen 5 Lea Vig McKinney N-7034 Trondheim Institute of Molecular Biology & Norway Physiology Tel: +47-73596093 University of Copenhagen E-mail: [email protected] Øster Farimagsgade 2A DK-1353 Copenhagen K Tiina Kuusela Denmark Department of Biological and Tel: +45 35322134 Environmental Sciences E-mail: [email protected] University of Helsinki Viikinkaari 1 Carsten Meier 00014 Helsinki Aresa Biodetection Finland Sølvgade 14 Tel: +358 9 191 59448 DK-1307 Copenhagen K E-mail: [email protected] Denmark Tel: +45 28356164 Andrea Lenk E-mail: [email protected] Department of Agricultural Sciences The Royal Veterinary and Agricultural Anders Laurell Blom Møller University Department of Agricultural Sciences Thorvaldsensvej 40 The Royal Veterinary and Agricultural DK-1871 Frederiksberg C University Denmark Thorvaldsensvej 40 Tel: +45 3528 3437 DK-1871 Frederiksberg C E-mail: [email protected] Denmark Tel: +45 3528 3465 Juri Lütje E-mail: [email protected] Institute of Molecular Biology & Physiology Per Muehlenbock University of Copenhagen Department of Botany Øster Farimagsgade 2A Stockholm University DK-1353 Copenhagen K Lilla Frescativaegen 5 Denmark SE-106 91 Stockholm Tel: +45 35322134 Sweden E-mail: [email protected] Tel: +46-8-163770 E-mail: [email protected] Frederikke Gro Malinovsky Institute of Molecular Biology & Markus Müller Physiology Department of Plant Physiology University of Copenhagen Umeå University Øster Farimagsgade 2A SE-90187 Umeå DK-1353 Copenhagen K Sweden Denmark Tel: +46 90 786 7888 Tel: +45 35322134 E-mail: [email protected] E-mail: [email protected] John Mundy Institute of Molecular Biology &

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

Physiology Norway University of Copenhagen Tel: +47 64 96 55 98 Øster Farimagsgade 2A E-mail: [email protected] DK-1353 Copenhagen K Denmark Elin Övernäs Tel: +45 35322131 Department of Physiological Botany E-mail: [email protected] Uppsala University Villavägen 6 Ragnhild Nesteskog SE-75236 Uppsala Dept. of Plant and Environmental Sciences Sweden University of Life Sciences Tel: +46 18 471 28 00 P.O.Box 5003 E-mail: [email protected] N-1432 Ås Norway Tapio Palva Tel: +47 64 96 55 98 Department of Biological and E-mail: [email protected] Environmental Sciences University of Helsinki Mari-Anne Newman Viikinkaari 5 Department of Plant Biology 00014 Helsinki The Royal Veterinary and Agricultural Finland University Tel: +358 9 191 59600 Thorvaldsensvej 40 E-mail: [email protected] DK-1871 Frederiksberg C Denmark Pai Pedas Tel: +45 3528 3303 Department of Agricultural Sciences E-mail: [email protected] The Royal Veterinary and Agricultural University Mads Eggert Nielsen Thorvaldsensvej 40 Carlsberg Research Center DK-1871 Frederiksberg C Gamle Carlsberg Vej 10 Denmark DK-2500 Valby Tel: +45 3528 3465 Denmark E-mail: [email protected] Tel: +45 33275370 E-mail: [email protected] Carsten Pedersen Department of Agricultural Sciences Lars Nilsson The Royal Veterinary and Agricultural Department of Physiological Botany University Uppsala University Thorvaldsensvej 40 Villavagen 6 DK-1871 Frederiksberg C SE-752 36 Uppsala Denmark Sweden Tel: +45 3528 3788 Tel: +46 18 1471 28 07 E-mail: [email protected] E-mail: [email protected] Jin-long Qiu Pernille Olsen Institute of Molecular Biology and Biosystems Department Physiology Risø National Laboratory Copenhagen University DK-4000 Roskilde Øster Farimagsgade 2A Denmark DK-1353 Copenhagen K Tel: +45 46 77 41 48 Denmark E-mail: [email protected] Tel: +45 35322135 E-mail: [email protected] Lene Olsen Dept. of Plant and Environmental Sciences Søren K. Rasmussen University of Life Sciences Department of Agricultural Sciences P.O.Box 5003 The Royal Veterinary and Agricultural N-1432 Ås University

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

Thorvaldsensvej 40 Fakultetsgatan 1 DK-1871 Frederiksberg C SE-70182 Örebro Denmark Sweden Tel: +45 3528 3436 Tel: + 46 19301034 E-mail: [email protected] E-mail: [email protected]

Verana Reiser Nikolai Scherbak Department of Plantbiochemistry and - Department of Natural Sciences physiology Örebro University University of Konstanz Fakultetsgatan 1 Universitätsstrasse 10 SE-70182 Örebro D-78457 Konstanz Sweden Germany Tel: + 46 19301034 Tel: +49-7531-882908 E-mail: [email protected] E-mail: [email protected] Desiree Schüssler Mikael Ristilä Department of Agricultural Sciences Department of Natural Sciences The Royal Veterinary and Agricultural Örebro University University Fakultetsgatan 1 Thorvaldsensvej 40 SE-701 82 Örebro DK-1871 Frederiksberg C Sweden Denmark Tel: +46 19301034 Tel: +45 3528 3419 E-mail: [email protected] E-mail: [email protected]

Odd Arne Rognli Carl-Johan Simola Department of Biology Department of Physiological Botany Norwegian University of Science and Uppsala University Technology Villavägen 6 Høyskoleringen 5 SE-75236 Uppsala N-7034 Trondheim Sweden Norway Tel: +46 18 471 28 07 Tel: +47-73596093 E-mail: [email protected] E-mail: [email protected] Eva Söderman Jens Rohloff Department of Physiological Botany Department of Biology Uppsala University Norwegian University of Science and Villavägen 6 Technology SE-75236 Uppsala Høyskoleringen 5 Sweden N-7034 Trondheim Tel: +46 18 471 28 20 Norway E-mail: [email protected] Tel: +47-73596093 E-mail: [email protected] Grethe-Elisabeth Stenvik Department of Molecular Biosciences Barbro E. Sæther University of Oslo Department of Molecular Biosciences P.O. Box 1041 Blindern University of Oslo N-0316 Oslo P.O. Box 1041 Blindern Norway N-0316 Oslo Tel: +47 22 85 45 71 Norway E-mail: [email protected] Tel: +47 22 854573 E-mail: [email protected] Floor ten Hoopen Department of Agricultural Sciences Helena Sävenstrand The Royal Veterinary and Agricultural Department of Natural Sciences University Örebro University Thorvaldsensvej 40

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005

DK-1871 Frederiksberg C Environmental Sciences Denmark University of Helsinki Tel: +45 3528 3465 Viikinkaari 1 E-mail: [email protected] 00014 Helsinki Finland Hans Thordal-Christensen Tel: +358 9 191 59440 Department of Agricultural Sciences E-mail: [email protected] The Royal Veterinary and Agricultural University Michael Wrzaczek Thorvaldsensvej 40 Department of Biol. & Environ. Sci. DK-1871 Frederiksberg C University of Helsinki Denmark Viikinkaari 1 Tel: +45 3528 3788 00014 Helsinki E-mail: [email protected] Finland Tel: +358 9 191 57 773 Sine Hovbye Topp E-mail: [email protected] Department of Agricultural Sciences The Royal Veterinary and Agricultural Ziguo Zhang University Department of Agricultural Sciences Thorvaldsensvej 40 The Royal Veterinary and Agricultural DK-1871 Frederiksberg C University Denmark Thorvaldsensvej 40 Tel: +45 3528 3788 DK-1871 Frederiksberg C E-mail: [email protected] Denmark Tel: +45 3528 3788 Diem Hong Tran E-mail: [email protected] Department of Biology Norwegian University of Science and Jian-Kang Zhu Technology Department of Botany and Plant Sciences Høyskoleringen 5 University of California N-7034 Trondheim Riverside Norway CA 92521 Tel: +47-73551280 USA E-mail: [email protected] Tel: +1 951 827 7117 E-mail: jian [email protected] Annikki Welling Department of Biological and

The 5th Workshop in the Denmark, Nordic Arabidopsis Network Oct. 13-15, 2005